Treatment or prophylaxis of proliferative conditions

ABSTRACT

The invention relates to novel compounds for use in the treatment or prophylaxis of cancers and other proliferative conditions that are for example characterized by cells that express cytochrome P450 1B1 (CYP1B1) and allelic variants thereof. The invention also provides pharmaceutical compositions comprising one or more such compounds for use in medical therapy, for example in the treatment of prophylaxis of cancers or other proliferative conditions, as well as methods for treating cancers or other conditions in human or non-human animal patients. The invention also provides methods for identifying novel compounds for use in the treatment of prophylaxis of cancers and other proliferative conditions that are for example characterized by cells that express CYP1 B1 and allelic variants thereof. The invention also provides a method for determining the efficacy of a compound of the invention in treating cancer.

FIELD

The present invention relates to novel compounds for use in thetreatment or prophylaxis of cancers and other proliferative conditionsthat are for example characterized by cells that express cytochrome P4501B1 (CYP1B1) and allelic variants thereof. The present invention alsoprovides pharmaceutical compositions comprising one or more suchcompounds for use in medical therapy, for example in the treatment ofprophylaxis of cancers or other proliferative conditions, as well asmethods for treating cancers or other conditions in human or non-humananimal patients. The present invention also provides methods foridentifying novel compounds for use in the treatment of prophylaxis ofcancers and other proliferative conditions that are for examplecharacterized by cells that express CYP1B1 and allelic variants thereof.The present invention also provides a method for determining theefficacy of a compound of the invention in treating cancer.

BACKGROUND

CYP1B1 is a member of the dioxin-inducible CYP1 gene family which alsoincludes CYP1A1 and CYP1A2 as described by Sutter et al. (J Biol. Chem.,May 6; 269(18):13092-9, 1994). CYP1B1 is a heme-thiolate mono-oxygenaseenzyme that is capable of metabolizing and activating a variety ofsubstrates including steroids, xenobiotics, drugs and/or prodrugs.CYP1B1 protein is expressed to a high frequency in a wide range ofprimary and metastatic human cancers of different histogenic types andis not expressed or at negligible levels in normal tissue. (see, e.g.:McFadyen M C, Melvin W T and Murray G I, “Cytochrome P450 Enzymes: NovelOptions for Cancer Therapeutics”, Mol Cancer Ther., 3(3): 363-71, 2004;McFadyen M C and Murray G I, “Cytochrome P450 1B1: a Novel AnticancerTherapeutic Target”, Future Oncol., 1(2): 259-63, 2005; Sissung T M,Price D K, Sparreboom A and Figg W D, “Pharmacogenetics and Regulationof Human Cytochrome P450 1B1: Implications in Hormone-Mediated TumorMetabolism and a Novel Target for Therapeutic Intervention”, Mol. CancerRes., 4(3):135-50, 2006).

More specifically, CYP1B1 has been shown to be expressed in bladder,brain, breast, colon, head and neck, kidney, lung, liver, ovarian,prostate and skin cancers, without being expressed in the correspondingnormal tissue. For example, Barnett, et al, in Clin. Cancer Res.,13(12): 3559-67, 2007, reported that CYP1B1 was over-expressed in glialtumours, including glioblastomas, anaplastic astrocytomas,oligodendrogliomas and anaplastic oligodendrogliomas, but not unaffectedbrain tissue; Carnell, et al., in Int. J. Radiat. Oncol. Biol. Phys.,58(2): 500-9, 2004, reported that CYP1B1 was over-expressed in prostateadenonocarcinomas, but not in matched normal prostate tissue; Carnell,et al., 2004 (ibid.) also showed that CYP1B1 is expressed in (n=22,100%) of bladder carcinomas; Downie, et al., in Clin. Cancer Res.,11(20): 7369-75, 2005 and McFadyen, et al., in Br. J. Cancer, 85(2):242-6, 2001, reported increased expression of CYP1B1 in primary andmetastatic ovarian cancer, but not in normal ovary tissue; and Gibson,et al., in Mol. Cancer Ther., 2(6): 527-34, 2003, and Kumarakulasingham,et al., in Clin. Cancer Res., 11(10): 3758-65, 2005, reported thatCYP1B1 was over-expressed in colon adenocarcionomas as compared tomatched normal tissue.

Several studies have shown that CYP1B1 is over-expressed in breastcancer as compared to matched normal tissue (see, e.g.: Murray G I,Taylor M C, McFadyen M C, McKay J A, Greenlee W F, Burke M D and MelvinW T, “Tumor-Specific Expression of Cytochrome P450 CYP1B1”, Cancer Res.,57(14): 3026-31, 1997; Haas S, Pierl C, Harth V, Pesch B, Rabstein S,Bruning T, Ko Y, Hamann U, Justenhoven C, Brauch H and Fischer H P,“Expression of Xenobiotic and Steroid Hormone Metabolizing Enzymes inHuman Breast Carcinomas”. Int. J. Cancer, 119(8): 1785-91, 2006; McKay JA, Murray G I, Ah-See A K, Greenlee W F, Marcus C B, Burke M D andMelvin W T, “Differential Expression of CYP1A1 and CYP1B1 in HumanBreast Cancer”, Biochem. Soc. Trans., 24(2): 327S, 1996).

Everett, et al., in J. Clin. Oncology, 25: 18S, 2007, reported thatCYP1B1 was over-expressed in malignant melanoma and disseminated diseasebut not in normal skin. Chang, et al., in Toxico. Sci., 71(1): 11-9,2003, reported that CYP1B1 protein is not present in normal liver butEverett, et al., 2007 (ibid.) confirmed CYP1B1 over-expression inmelanoma stage IV metastasis to the liver but not in the adjacent normalliver tissue.

Greer, et al., in Proc. Am. Assoc. Cancer Res., 45: 3701, 2004, reportedthat CYP1B1 was over-expressed during the malignant progression of headand neck squamous cell carcinoma but not in normal epithelium.

McFadyen, et al., in Br. J. Cancer, 91(5): 966-71, 2004, detected CYP1B1in renal carcinomas but not in corresponding normal tissue.

Murray, et al., 2004 (ibid.) used immunohistochemistry to showover-expression of CYP1B1 in lung cancer cells as compared to normallung tissue. Su, et al., in Anti-Cancer Res., 2, 509-15, 2009, usedimmunohistochemistry to show over-expression of CYP1B1 in advanced stageIV non-small cell lung cancer compared to earlier stages of the disease.

It is evident from the numerous disclosures cited above that CYP1B1expression is characteristic of a range of different cancers and otherproliferative conditions, and that CYP1B1 expression may be used todefine such a range of cancers and other conditions. As normal(non-cancerous) cells do not express significant levels of CYP1B1, itmay also be reasonably expected that compounds that exhibit cytotoxicityin cells expressing CYP1B1, but are substantially non-cytotoxic innormal cells, would have utility as targeted anti-cancer agents incancers characterized by CYP1B1 expression. By “targeted” is meant thatsuch compounds could be delivered systemically and would only beactivated in the presence of cancerous cells expressing CYP1B1,remaining substantially non-toxic to the rest of the body.

Furthermore, a number of cytochrome P450 enzymes are known to metaboliseand detoxify a variety of anticancer drugs. McFadyen, et al. n (BiochemPharmacol. 2001, Jul. 15; 62(2): 207-12) demonstrated a significantdecrease in the sensitivity of docetaxel in cells expressing CYP1B1 ascompared with non-CYP1B1 expressing cells. This finding indicates thatthe presence of CYP1B1 in cells may decrease their sensitivity to somecytotoxic drugs. CYP1B1-activated prodrugs may therefore be useful forthe treatment of cancers whose drug resistance is mediated by CYP1B1.

Furthermore, the CYP1B1 gene is highly polymorphic in cancer and severalsingle nucleotide polymorphisms contained within the CYP1B1 gene havebeen identified that alter the expression and/or activity of the encodedprotein. Of these, the CYP1B1*3 (4326C>G; L432V) allele has beencharacterized by both increased expression and enzyme kinetics of CYP1B1toward several substrates as described by Sissung, et al. in Mol CancerTher., 7(1): 19-26, 2008 and references quoted therein. This findingindicates that not only CYP1B1 but the allelic variants of the enzymemay also contribute to prodrug activation and cancer targeting.

Prodrugs have been investigated as a means to lower the unwantedtoxicity or some other negative attribute of a drug without loss ofefficacy. A prodrug is a drug that has been chemically modified torender it inactive but that, subsequent to administration, ismetabolized or otherwise converted to an active form of the drug in thebody. The over-expression of CYP1B1 in primary tumours and metastaticdisease compared to normal tissue offers a tremendous opportunity forthe development of CYP1B1-activated prodrugs for targeted cancer therapyas reviewed by McFadyen et al., Mol Cancer Ther., 3(3), 363-71, 2004.Indeed, the discovery and development of CYP1B1-activated prodrugs fortargeted cancer therapy is likely to offer significant pharmacologicaladvantages over existing non-targeted cytochrome P450-activated prodrugsused clinically such as the prodrug alkylating agents cyclophosphamide,ifosfamide, dacarbazine, procarbazine which are activated by cytochromeP450s expressed in normal tissue as reviewed by Patterson L H and MurrayG I in Curr Pharm Des., 8(15): 1335-47, 2002.

The human cytochrome P450 family contains 57 active isozymes, whichfunction in normal metabolism, influence drug pharmacokinetics andeffect negative outcomes in patients through drug-drug interactions. Thecytochrome P450 isoenzymes metabolize approximately two thirds of knowndrugs in humans, with 80% of this attributable to five isozymes, namelyCYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 as described in Ortiz deMontellano, PR (ed.) Cytochrome P450: structure, mechanism, andbiochemistry, Kluwer Academic/Plenum Publishers, New York, 2005.

Among the genes discovered by intiatives in the human genome project areCYP2R1, CYP2W1, CYP2S1, CYP2S1, CYP2U1 but the function, polymorphismand regulation of these genes are still to be fully elucidated asreviewed by Ingelman-Sundberg, M., Toxicol. Appl. Pharmacol., 207, 52-6,2005. In addition to CYP1B1 a number of these cytochrome P450oxidoreductases are extrahepatic and over-expressed in cancer. Severalcytochrome P450s including CYP1B1, CYP2A/2B, CYP2F1, CYP2R1, CYP2U1,CYP3A5, CYP3A7, CYP4Z1, CYP26A1, and CYP 51 are present at asignificantly higher level of intensity than in normal ovary asdetermined by immunohistochemistry and light microscopy, as described byDownie et al., Clin. Cancer Res., 11(20): 7369-75, 2005. Furthermore,using similar methods of detection in primary colorectal cancer, severalcytochrome P450s, including CYP1B1, CYP2S1, CYP2U1, CYP3A5, and CYP51,are frequently over-expressed compared to normal colon as descried byKumarakulasingham et al, Clin. Cancer Res., 11(10): 3758-65, 2005. Inthe same study several cytochrome P450s, including CYP1B1, CYP2A/2B,CYP2F1, CYP4V2, and CYP39, correlated with their presence in the primarytumour. CYP2W1 has also been shown to be over-expressed in colorectalcancer according to Elder et al, Eur. J. Cancer, 45(4): 705-12. CYP4Z1is over-expressed in breast carcinoma is a gene associated withnon-small cell lung cancer promotion and progression as described byReiger et al., Cancer Res., 64(7): 2357-64, 2004 and Bankovic et al.,Lung Cancer, 67(2): 151-9, 2010, respectively.

A major challenge in the field is elucidation of the function of humancytochrome P450s of so-called ‘orphan’ status with unknown substratespecificity as reviewed by Strak K and Guengerich F P in Drug Metab.Rev., 39(2-3): 627-37, 2007. A number of substrates are known for CYP1B1few of which are specifically metabolised by the enzyme, for example7-ethoxyresorufin undergoes oxidative de-ethylation when activated byall members of the CYP1 family, including CYP1A1, CYP1A2, and CYP1B1, asdescribed by Chang T K and Waxman D J in Method Mol. Biol., 320, 85-90,2006. A number of fluorgenic and luminogenic probe substrates areavailable to assess cytochrome P450 activity with high sensitivity butthey exhibit broad specificity and as such are metabolised by a range ofcytochrome P450 enzymes in the CYP1, CYP2, and CYP3 families. Forexample, Cali et al., Expert Opin. Drug Toxicol., 2(4): 62-45. 2006describes the use of luminogenic substrates which couple to fireflyluciferase luminescence in a technology called P450-Glo. Anotherexample, is 7-ethoxycoumarin which undergoes cytochrome P450-catalyzed7-ethoxycoumarin O-deethylation to release the highly fluourescent anionas described by Waxman D J and Change T K H in “The use of7-ethoxycoumarin to minitor multiple enzymes in the human CYP1, CYP2,CYP3 families” in Methods in Molecular Biology, vol. 320, CytochromeP450 Protocols, Second Edition, edited by Phillips I R and Shephard, EA, 2006.

Everett et al., Biochem. Pharmacol., 63, 1629-39, 2002 describe thereductive fragmentation of model indolequinone prodrugs by cytochromeP450 reductase (not to be confused with cytochrome P450s) in anoxia torelease the 7-hydroxy-4-methylcoumarin anion. The model prodrug wasnon-fluorescent at the pre-selected emission wavelength and reductivefragmentation could be accurately measured by monitoring the productionof the coumarin anion (λ_(ex)=380 nm/λ_(em)=450 nm) using kineticspectrofluorimetry.

Interactions between a limited number of compounds (typically <100) andcytochrome P450s isozymes have been described but results from suchstudies are difficult to compare because of the differences intechnologies, assay conditions and data analysis methods as described byRendic, S. “Summary of information on human CYP enzymes: human P450metabolism data” in Drug Metab. Rev., 34, 83-448, 2002. Mnaycomputational strategies have been advanced to generate predictivecytochrome P450 isozyme substrate activity models but these are limitedby a lack of a single large, diverse data set of cytochrome P450 isozymeactivities as described by Veith et al., Nature Biotechnology, 27,1050-55, 2009. The authors describe the construction of cytochrome P450bioactivity databases using quantitative high-throughput screening (HTS)with a bioiluminescent enzyme substrate inhibition assay to screen17,143 chemical compounds against five cytochrome P450 isozymes (CYP1A2,2C9, 2C19, 2D6, and 3A4) expressed in normal tissues mainly the liverand responsible for so-called phase 1 metabolism of drugs. It wasconcluded that the database should aid in constructing and testing newpredictive models for cytochrome P450 activity to aid early stage drugdiscovery efforts.

Jensen et al., J. Med. Chem., 50, 501-11, 2007 describe the methods forthe in silico prediction of CYP2D6 and CYP3A4 inhibition based on anovel Gaussian Kernel weighted k-nearest neighbour (k-NN) algorithmbased on Tanimoto similarity searches on extended connectivityfingerprints. The data set included modelling of 1153 and 1182 drugcandidates tested for CYP2D6 and CYP3A4 inhibition in human livermicrosomes. For CYP2D6, 82% of the classified test compounds werepredicted to the correct class and CYP3A4, 88% of the classified testcompounds were correctly classified.

Theoretically it may be possible to use cytochrome P450 HTS to build alarge database of bioactivities for tumour and normal tissue cytochromeP450s and then develop a substrate prediction model as a basis for thedesign and synthesis of selective CYP1B1-activated prodrugs whilescreening out for pharmacological liabilities associated with Phase 1metabolism by normal tissue cytochrome P450s. However, the reduction topractice is not obvious from prior art and has to be rationalisedagainst prodrug structure and mechanism of conversion to the active drugwhen activated by tumour-expressing cytochrome P450s.

Utilization of so-called ‘trigger-linker-effector’ chemistry in prodrugdesign requires the activation of the trigger to initiate thefragmentation of a linker to release an effector (typically an activedrug), the biological activity of which is masked in the prodrug form.The modular design of selective prodrugs targeted at tumour-expressingcytochrome P450s such as CYP1B1 require (1) the identification ofselective trigger moieties, (2) the use of bio-stable linkers whichfragment efficiently following trigger activation (usually by aromatichydroxylation), and (3) suitable effectors or drugs which do notinterfere with the efficiency of the triggering process.

CYP1B1 mRNA is expressed constitutively in all normal extrahepatic humantissues, though the protein is usually undetectable. In contrast, CYP1B1protein is expressed at high levels in tumours. It is understood thatfor a large range of established or immortilized tumour cell lines (suchas the MCF-7 breast cancer cells) originating from humans which haveundergone significant passaging in vitro but does not constitutivelyexpress active CYP181 protein. Although CYP1B1 is not constitutivelyexpressed in MCF-7 breast tumour cells it is possible to induce CYP1enzyme expression both at the mRNA and protein level by treating witharyl hydrocarbon agonists such as the dioxin TCDD.

WO 99/40944 describes prodrugs that comprise a drug moiety bound to acarrier framework, the prodrug being described activated as thoughhydroxylation by CYP1B1 to release the drug moiety.

SUMMARY

We have surprisingly found that the compounds described herein, distinctover those described in WO 99/40944, are broken down in certain cells,in particular those that express cytochrome P450 1B1 (hereinafterCYP1B1), but not in normal cells, as a consequence of the compoundscollapsing upon hydroxylation (e.g. effected by CYP1B1-expressingcells), and in particular by cancerous cells.

According to a first aspect therefore the present invention provides acompound of formula (I):

(wherein:

X¹ is such that —X—X² is —O—X², —S—X², —SO₂—O—X², —SO₂NZ¹⁰—X²,conjugated alkenemethyloxy, conjugated alkenemethylthio, conjugatedalkenemethylSO₂—O, conjugated alkenemethyl-SO₂NZ¹⁰ or of the formula:

—X² is absent or is such that X¹—X²-Effector is one of

each n and m is independently 0 or 1;

p is 0, 1 or 2;

X³ is oxygen or sulfur and additionally, when m=0, may be SO₂—O,SO₂NZ¹⁰, conjugated alkenemethyloxy, conjugated alkenemethylthio,conjugated alkenemethyl-SO₂—O or conjugated alkenemethyl-SO₂NZ¹⁰;

each of Y¹, Y² and Y³ is independently carbon or nitrogen, wherein if Y¹is nitrogen, Z¹ is absent, if Y² is nitrogen, Z³ is absent and if Y³ isnitrogen, Z⁵ is absent;

Y⁴ is an oxygen, carbon or nitrogen atom, sulfoxide or sulfone;

—Y⁵— is either (i) a single bond, (ii)=CH—, wherein the double bond = in═CH— is connected to Y⁴, or (iii) —CH₂— or —CH₂CH₂—, or one of (ii) to(iii) wherein the hydrogen atom in (ii) is or one or more hydrogen atomsin (iii) are replaced with a substituent Z¹¹, wherein Z¹¹ is selectedindependently from alkyl, alkenyl, alkynyl, aryl, aralkyl, alkyloxy,alkenyloxy, alkynyloxy, aryloxy, aralkyloxy, alkylthioxy, alkenylthioxy,alkynylthioxy, arylthioxy, aralkylthioxy, amino, hydroxy, thio, halo,carboxy, formyl, nitro and cyano;

each of Z¹—Z⁴, where present, are independently selected from hydrogen,alkyl, alkenyl, alkynyl, aryl, aralkyl, alkyloxy, alkenyloxy,alkynyloxy, aryloxy, aralkyloxy, alkylthioxy, alkenylthioxy,alkynylthioxy, arylthioxy, aralkylthioxy, amino, hydroxy, thio, halo,carboxy, formyl, nitro and cyano; and Z⁵, where present, isindependently selected from hydrogen alkyl, alkenyl, alkynyl, aryl,aralkyl, alkyloxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy,alkylthioxy, alkenylthioxy, alkynylthioxy, arylthioxy, aralkylthioxy,amino, hydroxy, thio, carboxy, formyl, nitro and cyano, or one of Z² &Z³, Z³ & Z⁴ and Z⁴ and Z⁵ together with the atoms to which they areconnected form an aromatic ring fused to the remainder of the compound,provided that at least one of Z¹, Z² and Z⁴ is hydrogen;

Z⁶ is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl and aralkyl;

none, one or two of Y⁶ may be nitrogen atoms with the remainder beingcarbon atoms;

each Z⁷ is independently hydrogen, alkyl or aryl;

each Z⁸ is independently selected from hydrogen, an electron withdrawinggroup, unsubstituted C₁-C₆ alkyl, substituted C₁-C₆ alkyl, unsubstitutedC1-C₆ alkoxy, and substituted C₁-C₆ alkoxy where the substituted alkylor alkoxy are substituted with one or more groups selected from ether,amino, mono- or di-substituted amino, cyclic C₁-C₅ alkylamino,imidazolyl, C₁₋₆ alkylpiperazinyl, morpholino, thiol, thioether,tetrazole, carboxylic acid, ester, amido, mono- or di-substituted amido,N-connected amide, N-connected sulfonamide, sulfoxy, sulfonate,sulfonyl, sulfoxy, sulfinate, sufinyl, phosphonooxy, phosphate andsulfonamide;

each Z⁹ is independently oxygen or sulfur;

Z¹⁰ is hydrogen or alkyl, for example a C₁₋₄ alkyl;

Effector is a molecule having a pharmacological, diagnostic or screeningfunction),

or a pharmaceutically acceptable salt, ester, amide or solvate thereof.

Viewed from a second aspect, the invention provides a compositioncomprised of a compound according to the first aspect of the invention,or a pharmaceutically acceptable salt, ester, amide or solvate thereof,together with a pharmaceutically acceptable carrier.

Viewed from a third aspect the invention provides a compound accordingto the first aspect of the invention, or a pharmaceutically acceptablesalt, ester, amide or solvate thereof, for use as a medicament.

Viewed from a fourth aspect, the invention provides a compound accordingto the first aspect of the invention, or a pharmaceutically acceptablesalt, ester, amide or solvate thereof, for use in a method of treatmentor prophylaxis of a proliferative condition.

Viewed from a fifth aspect, the invention provides a method of treatmentor prophylaxis of a proliferative condition, said method comprisingadministering a therapeutically or prophylactically useful amount of acompound according to the first aspect of the invention, orpharmaceutically acceptable salt, ester, amide or solvate thereof, to asubject in need thereof.

Viewed from a sixth aspect, the invention provides the use of a compoundaccording to the first aspect of the invention or a pharmaceuticallyacceptable salt, ester, amide or solvate thereof, for the preparation ofmedicament for use in a method of treatment or prophylaxis of aproliferative condition.

Viewed from a seventh aspect, the invention provides a method ofidentifying a compound that is specifically activated by a cytochromeP450 enzyme, said method comprising the steps of:

(a) contacting a set of compounds, according to the first aspect of theinvention in which Effector is a fluorophore, with said cytochrome P450enzyme and determining if said contact results in release of saidfluorophore from one or more compounds of said set;

(b) contacting said set of compounds with a control tissue, tissue orcell extract, or enzyme and determining if said contact results inrelease of said fluorophore from one or more compounds of said set; and

(c) identifying said compound specifically activated by said cytochromeP450 as any compound in said set of compounds that releases saidfluorophore in step (a) but not, or only to a much lesser extent, instep (b).

Viewed from an eighth aspect, the invention provides a method fordetermining whether a compound of the invention, wherein Effector is amolecule having a pharmacological function, is efficacious in treatingcancer, said method comprising administering said compound to an animalhaving cancer, wherein said cancer is resultant from implantation ofeither a recombinant cell modified so as to express constitutively acytochrome P450 enzyme, a tissue taken directly from a tumor or acancer, or a cell from an early passage cell line derived from a tissuetaken directly from a tumor or a cancer that expresses said cytochromeP450 enzyme at levels similar to those from the tumor or cancer fromwhich it originates.

Further aspects and embodiment of the invention will follow from thediscussion that follows below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts Western blots showing the detection of CYP1B1 expressionin a transfected CHO/CYP1B1/CPR cell line (panel A) and CYP1A1expression in a transfected CHO/CYP1A1/CPR cell line (panel B). Detailsare provided in the experimental section below.

FIG. 2a shows a mechanism for CYP1B1-induced 3-hydroxylation of acompound of the invention (referred to herein as SU025-04) followed byspontaneous release of a cytotoxic Effector molecule(N,N′-bis(2-chloroethyl)phosphorodiamidate (also known as IPM chloride)by 1,4 elimination.

FIG. 2b shows a mechanism for CYP1B1-induced 4-hydroxylation of acompound of the invention (referred to herein as SU025-04) followed byspontaneous release of a cytotoxic Effector molecule(N,N′-bis(2-chloroethyl)phosphordiamidate (also known as IPM chloride)by 1,6 elimination.

FIG. 2c shows a mechanism for CYP1B1-induced 6-hydroxylation of acompound of the invention (referred to herein as SU025-04) followed byspontaneous release of a cytotoxic Effector molecule(N,N′-bis(2-chloroethyl)phosphordiamidate (also known as IPM chloride)by 1,8 elimination.

FIG. 3 shows a mechanism for CYP1B1-induced 6-hydroxylation of acompound of the invention (referred to herein as SU024-1-03) followed byspontaneous release of an Effector molecule by 1,8 elimination.

DETAILED DESCRIPTION

The present invention arises from the provision of prodrugs in which aso-called Effector molecule, which may be a cytostatic, cytotoxic,diagnostic or screening molecule as described in greater detailhereinafter, is chemically modified by reacting it whereby to form acompound of formula (I). We have found that hydroxylation of compoundsof formula (I), in particular CYP1B1-induced hydroxylation, allowsrelease of the Effector molecules by a collapse of the compounds offormula (I) which happens spontaneously upon direct hydroxylation orhydroxylation via epoxide formation.

In overview, the structure of the compounds of formula (I) may beconsidered to comprise three parts: a trigger region, a linker and anEffector molecule. The trigger serves as a substrate for the typicallyCYP1B1-induced hydroxylation and may be generally understood to comprisethe bicyclic moiety depicted on the left hand side of formula (I) andthe substituents thereof, i.e. comprising that part of the compoundscontaining Y¹—Y⁵, Z¹—Z⁶ and the remaining carbon atoms to which some ofthese moieties are attached. The trigger region of the compounds isattached through a linking region comprising the C(Z⁷)—X¹—X² unit to theEffector molecule which is labelled as such.

The make-up and variability of these three regions—the trigger, linkerand Effector regions—of the compounds of formula (I) are now described.

In the discussion that follows, reference is made to a number of terms,which are to be understood to have the meaning provided, below, unlessthe context dictates to the contrary.

By alkyl is meant herein a saturated hydrocarbyl radical, which may bestraight-chain, cyclic or branched (typically straight-chain unless thecontext dictates to the contrary). Where an alkyl group has one or moresites of unsaturation, these may be constituted by carbon-carbon doublebonds or carbon-carbon triple bonds. Where an alkyl group comprises acarbon-carbon double bond this provides an alkenyl group; the presenceof a carbon-carbon triple bond provides an alkynyl group. Typicallyalkyl, alkenyl and alkynyl groups will comprise from 1 to 25 carbonatoms, more usually 1 to 10 carbon atoms, more usually still 1 to 6carbon atoms it being of course understood that the lower limit inalkenyl and alkynyl groups is 2 carbon atoms and in cycloalkyl groups 3carbon atoms.

Alkyl, alkenyl or alkynyl groups may be substituted, for example once,twice, or three times, e.g. once, i.e. formally replacing one or morehydrogen atoms of the alkyl group. Examples of such substituents arehalo (e.g. fluoro, chloro, bromo and iodo), aryl hydroxy, nitro, amino,alkoxy, alkylthio, carboxy, cyano, thio, formyl, ester, acyl, thioacyl,amido, sulfonamido, carbamate and the like.

By carboxy is meant herein the functional group CO₂H, which may be indeprotonated form (CO₂ ⁻).

Halo is fluoro, bromo, chloro or iodo.

By acyl and thioacyl are meant the functional groups of formulae—C(O)-alkyl or —C(S)-alkyl respectively, where alkyl is as definedhereinbefore.

By ester is meant a functional group comprising the moiety —OC(═O)—.

By amido is meant a functional group comprising the moiety —N(H)C(═O)—;by carbamate is meant a functional group comprising the moiety—N(H)C(═O)O—; and by sulfonamido is meant a functional group comprisingthe moiety —SO₂N(H)₂—, in which each hydrogen atom depicted may bereplaced (independently in sulfonamido) with alkyl or aryl.

Alkyloxy (synonymous with alkoxy) and alkylthio moieties are of theformulae —O-alkyl and —S-alkyl respectively, where alkyl is as definedhereinbefore.

Likewise alkenyloxy, alkynyloxy, alkenylthio and alkynylthio are of theformulae —O-alkenyl, -Oalkynyl, -Salkenyl and Salkynyl, where alkenyland alkynyl are as defined hereinbefore.

By amino group is meant herein a group of the formula —N(R)₂ in whicheach R is independently hydrogen, alkyl or aryl, e.g. an unsaturated,unsubstituted C₁₋₆ alkyl such as methyl or ethyl, or in which the two Rsattached to the nitrogen atom N are connected. One example of this iswhereby —R—R— forms an alkylene diradical, derived formally from analkane from which two hydrogen atoms have been abstracted, typicallyfrom terminal carbon atoms, whereby to form a ring together with thenitrogen atom of the amine. As is known the diradical in cyclic aminesneed not necessarily be alkylene: morpholine (in which —R—R— is—(CH₂)₂O(CH₂)₂—) is one such example from which a cyclic aminosubstituent may be prepared.

References to amino herein are also to be understood as embracing withintheir ambit quaternised or protonated derivatives of the aminesresultant from compounds comprising such amino groups. Examples of thelatter may be understood to be salts such as hydrochloride salts.

By aryl is meant herein a radical formed formally by abstraction of ahydrogen atom from an aromatic compound.

Arylene diradicals are derived from aromatic moieties, formally, byabstraction of two hydrogen atoms, and may be and typically are, unlessthe context specifically dictates to the contrary, monocyclic, forexample, phenylene. As known to those skilled in the art, heretoaromaticmoieties are a subset of aromatic moieties that comprise one or moreheteroatoms, typically O, N or S, in place of one or more carbon atomsand any hydrogen atoms attached thereto. Exemplary heteroaromaticmoieties, for example, include pyridine, furan, pyrrole and pyrimidine.Further examples of heteroaromatic rings include pyrdidyl, pyridazine(in which 2 nitrogen atoms are adjacent in an aromatic 6-membered ring);pyrazine (in which 2 nitrogens are 1,4-disposed in a 6-membered aromaticring); pyrimidine (in which 2 nitrogen atoms are 1,3-disposed in a6-membered aromatic ring); or 1,3,5-triazine (in which 3 nitrogen atomsare 1,3,5-disposed in a 6-membered aromatic ring).

Aryl or arylene radicals may be substituted one or more times with anelectron-withdrawing group (for example a group selected from halo,cyano (—CN), haloalkyl, amide, nitro, keto (—COR), alkenyl, alkynyl,quarternary amino (—N⁺R₃), ester, amido (—CONR₂), N-connected amido(—NR—C(═O)—R), N-connected sulfonamido (—NR—S(═O)₂R), sulfoxy(—S(═O)₂OH), sulfonate (S(═O)₂OR), sulfonyl (S(═O)₂R) and sulfonamide(—S(═O)₂—NR₂), where (each) R is independently selected from a C₁-C₆alkyl group), a C₃-C₂₀ heterocyclic group, or a C₃-C₂₀ aryl group,typically a C₁-C₆ alkyl group, unsubstituted C₁-C₆ alkoxy, andsubstituted C₁-C₆ alkoxy where the substituted alkyl or alkoxy aresubstituted with one or more groups selected from ether, amino, mono- ordi-substituted amino, cyclic C₁-C₅ alkylamino, imidazolyl, C₁-C₆alkylpiperazinyl, morpholino, thiol, thioether, tetrazole, carboxylicacid, ester, amide, mono- or di-substituted amide, N-connected amide(—NR—C(═O)—R), N-connected sulfonamide (—NR—S(═O)₂—R), sulfoxy(—S(═O)₂OH), sulfonate (S(═O)₂OR), sulfonyl (S(═O)₂R), sulfoxy(S(═O)OH), sulfinate (S(═O)OR), sulfinyl (S(═O)R),phosphonooxy(—OP(═O)(OH)₂), phosphate (OP(═O)(OR)₂), and sulfonamide(—S(═O)₂—NR₂), where in (each) R is independently selected from a C₁-C₆alkyl group, a C₃-C₂₀ heterocyclic group, or a C₃-C₂₀ aryl group.

The trigger region of the compounds of formula (I) generally comprises abicyclic moiety comprising an aromatic ring (that comprises the Y² andY³ moieties as indicated) fused to a second ring (that comprises the Y¹,Y⁴ and Y⁵ moieties that may be aromatic or non-aromatic.

Without being bound by theory, it is believed that the activity of thecompounds of formula (I) as substrates for hydroxylation, e.g. effectedby CYP1B1, is achieved in part by the structure of the trigger moietybeing susceptible to hydroxylation when Z² or Z⁴ is hydrogen, or whenY¹—Z¹ is C—H, the hydroxylation thus taking place at one of the threecarbon atoms of those to which Z² and Z⁴ are connected, and Y¹, where Y¹is carbon. As is depicted in FIG. 2, hydroxylation at any of thesepositions in a representative compound of the invention, labelledSU025-04, leads to spontaneous collapse of the compound by anelimination process, either a 1,4-, a 1,6- or a 1,8-elimination,depending upon at which of these positions hydroxylation takes place.

It will be noted from the structure of the compounds of formula (I)that, by virtue of the conjugation of carbon atoms to which Z² and Z⁴are attached through Y¹ to the linker moiety, that any of the threemechanisms for spontaneous breakdown of the compound may take placeindependently of the nature of the Z⁶—Y⁴—Y⁵ region of the compounds.Thus a wide variety to the nature of this region of the compounds offormula (I) may be tolerated as discussed below. Also, continuation ofthe region of conjugation is achieved inter alia by the use of theconjugated X¹ moieties described herein.

In the compounds of formula (I), each of the atoms indicated by Y¹, Y²and Y³ may independently be a carbon atom or a nitrogen atom. Where theatom concerned is a nitrogen atom, the respective substituent (Z¹, Z³ orZ⁵ respectively) will be absent. In certain embodiments of the inventionY² or Y³ is a carbon atom. In particular embodiments of the inventionboth Y² and Y³ are carbon atoms. According to either of theseembodiments—that in which both Y² or Y³ is a carbon atom or in which Y²and Y³ are carbon atoms—or in which neither Y² or Y³ is a carbon atom,Y¹ may be a carbon atom.

The substituents Z¹, Z² and Z⁴ may be generally as described in claim 1.However, at least one of these moieties is a hydrogen atom so as toallow a site for hydroxylation of the compound. In some embodiments ofthe invention either Z² or Z⁴ is hydrogen. In other embodiments Z² andZ⁴ is hydrogen. In either of these embodiments—that in which Z² or Z⁴ isa hydrogen atom or in which both Z² and Z⁴ are hydrogen atoms—or inwhich neither Z² or Z⁴ is a hydrogen atom, Z¹ may be hydrogen. Incertain embodiments of the invention each of Z¹, Z² and Z⁴ is a hydrogenatom.

Either Z³ or Z⁴ may, together with the adjacent substituent on thearomatic ring (i.e. Z² or Z⁴, or Z³ or Z⁵ respectively) may, togetherwith the atoms of the aromatic ring to which these substituents areconnected form an aromatic ring fused to the remainder of the compound.Thus, Z² and Z³, together with the carbon atom to which Z² is connected,and Y², may form an aromatic ring. Similarly, for example, Z⁴, Z⁵ andthe carbon atom to which Z⁴ is connected, and Y³, may together form anaromatic ring.

In certain embodiments of the invention, none or only two of the pairsof substituents Z² & Z³, Z³ & Z⁴ and Z⁴ & Z⁵ together form a fusedaromatic ring. Thus, in certain embodiments there are no aromatic ringsfused to the aromatic ring comprising Y² & Y³.

Specifically, substituents Z³ and Z⁵ are typically not part of anaromatic ring fused to the remainder of the compound of formula (I).Where this is the case, i.e. where these moieties are individualsubstituents, Z³ may be alkyl, alkenyl, alkynyl, aryl, aralkyl,alkyloxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy, alkylthioxy,alkenylthioxy, alkynylthioxy, arylthioxy, aralkylthioxy, amino, hydroxy,thio, halo, carboxy, formyl, nitro and cyano and Z⁵ may be alkyl,alkenyl, alkynyl, aryl, aralkyl, alkyloxy, alkenyloxy, alkynyloxy,aryloxy, aralkyloxy, alkylthioxy, alkenylthioxy, alkynylthioxy,arylthioxy, aralkylthioxy, amino, hydroxy, thio, carboxy, formyl, nitroand cyano. In certain embodiments of the invention, Z³ may be alkyl,alkenyl, alkynyl, aryl, aralkyl, alkyloxy, alkenyloxy, alkynyloxy,aryloxy, aralkyloxy, alkylthioxy, alkenylthioxy, alkynylthioxy,arylthioxy, aralkylthioxy, amino, hydroxy, thio, halo, carboxy, formyl,nitro and cyano.

In certain embodiments of the invention, Z³ and Z⁵ are individualsubstituents other than hydrogen atoms. Where Z³ and Z⁵ are the samesubstituent or otherwise, Z³ and Z⁵ according to certain embodiments ofthe invention are electron-donating groups such as alkoxy, alkylthioxy,aryloxy, arylthioxy. In particular embodiments of the invention, Z³ orZ⁵ are both amino or alkoxy, for example, C₁-C₆ alkoxy. Examples of suchalkoxy groups include methoxy, ethoxy, isopropoxy, n-propoxy and thelike. In certain embodiments of the invention either Z³ or Z⁵, or Z³ andZ⁵, are methoxy. In certain embodiments of this invention Z³ and Z⁵ arethe same and are any of the immediately aforementioned substituents, orclasses of substituent. As noted above, the compounds of formula (I) maybe varied significantly in their structure in the portion that comprisesZ⁶—Y⁴—Y⁵. Thus Y⁴ may be oxygen, sulfur, sulfoxide or sulfone whereuponthere is no Z⁶ substituent present (p=0), nitrogen (wherein p=0 or 1) ora carbon atom whereupon p=1 or 2. In certain embodiments of theinvention p=0 and Y⁴ is oxygen, sulfur, sulfone or sulfoxide. Inparticular embodiments of the invention p=0 and Y⁴ is oxygen or sulfur.In certain embodiments of the invention p=0 and Y⁴ is oxygen.

—Y⁵— may be one of (i) a single bond, in which case the trigger moietyis based upon the 6-membered aromatic Y²— and Y³-containing ring fusedto a 5-membered ring since in this embodiment Y⁵ is effectively absent;or (ii)=CH— in which the double bond = is connected to Y⁴. In theseembodiments of the invention the trigger moiety is thus made up of twofused aromatic rings and the skilled person will appreciate that, where—Y⁵— is ═CH— then Y⁴ is either a nitrogen atom and p=0 or a carbon atomand p=1. Finally, —Y⁵— may be (iii) —CH₂— or —CH₂CH₂— in which case thetrigger moiety comprises a bicyclic system comprising a 6- or 7-memberedring fused to the aromatic 6-membered ring substituted with Y² and Y³.In certain embodiments of the invention the or one or more of hydrogenor the hydrogen atoms specified in options (ii) and (iii) for —Y⁵— maybe replaced with a Z¹¹ moiety, for example an alkyl or halo moiety. Incertain embodiments of the invention no Z¹¹ is present. In particularembodiments of the invention —Y⁵— is a single bond, for example whereinp=0 and Y⁴ is oxygen, sulfur, sulfone or sulfoxide, p=0 and Y⁴ is oxygenor sulfur and in particular wherein p=0 and Y⁴ is oxygen.

The linking moiety CH(Z⁷)—X¹—X² is now described.

Z⁷ is hydrogen or an alkyl or aryl group, which, in certain embodimentsof the invention is unsubstituted. In certain embodiments of theinvention, the or each Z⁷ is an alkyl group, e.g. an unsubstituted alkylgroup such as an unsubstituted C₁-C₆ alkyl group. Examples of Z⁷moieties include methyl and ethyl. In particular embodiments of theinvention Z⁷=hydrogen such that—CH(Z⁷)— is methylene. In otherembodiments the or each Z⁷ moiety is a substituted alkyl group, e.g. asubstituted methyl or ethyl group. Examples of such embodiments includeamino-substituted alkyl groups, e.g. morpholino or piperidinyl alkylgroups, or other groups that confer enhanced water solubility.Alternatively the, each, or at least one Z⁷ may be an optionallysubstituted heteroaryl moiety such as pyridyl.

X¹ may be a variety of linking atoms or divalent linking moieties, forexample, X¹ may be oxygen, sulfur, sulfonamide or sulfonate ester. Inaddition, X¹ may be ethane-1,2-diylbis(methylcarbamate) or a conjugatedalkenemethyloxy moiety.

By a conjugated alkenemethyloxy moiety is meant a moiety of the formula(═CH—CH)_(q)═CH—CH₂—O— wherein q is an integer from 0 to 6, for examplefrom 0 to 3, e.g. 0 or 1. The skilled person will understand that theoxygen atom depicted in the alkenemethyloxy moieties may be substitutedwith a sulfur atom SO₂—O or SO₂NZ¹⁰ moiety, whereby to provideconjugated alkenemethyl sulfonate or conjugated alkenemethyl sulfonamidemoieties as recited hereinbefore in which the oxygen or sulfur atoms, orsulfonate of sulfonamide moieties (SO₂—O and SO₂—NZ¹⁰) are attached toX² or, if this is absent, Effector.

According to certain embodiments of the invention X¹ is oxygen orsulfur. In many embodiments of the invention X¹ is oxygen.

X² is an optional additional linking moiety, which is either absent orinterposed between X¹ and the Effector moiety.

X² may be comprised of a variety of moieties as described herein or maybe absent. In certain embodiments of the invention X² is absent orX¹—X²-Effector is one of

For example, X² may comprise an arylene-CH(Z⁷)X³ moiety (hereinafter—ArCH(Z⁷)X³— moiety) and/or an amide moiety. Where present, the—Ar—CH(Z⁷)X³— moiety may be flanked by one or two amide or thioamidegroups (C(Z⁹)NH). If flanked by one amide or thioamide group, this maybe disposed directly between the X¹ moiety and the aromatic ring of the—Ar—CH(Z⁷)X³ (wherein n=1) moiety or interposed between X³ and theEffector moiety (wherein m=1). Alternatively, an amide or thioamidegroup may be present in both or neither of these positions. In certainembodiments of the invention n=0 and m=1. When X² comprises a—Ar—CH(Z⁷)X³— moiety, whether or not this is flanked by one or two amideor thioamide moieties, the X¹ moiety that is attached to the aromaticring either directly or indirectly through an amide or thioamide moietymay be attached at either of the two positions in the aromatic ring thatare ortho to the CH(Z⁷)X³ moiety of the —Ar—CH(Z⁷)X³ system or at thepara position. Engineering these points of attachment in the aromaticrings of the X² moieties that comprise Ar—CH(Z⁷)X³— moieties permits1,4-, 1,6- or 1,8-elimination of the Effector molecule. It will beunderstood that the arylene group present in certain embodiments of X²may be heteroaromatic, that is to say one or two or atoms Y⁶ may benitrogen atoms with the remainder being carbon atoms. An example of sucha heteroarylene moiety is pyridylene, in which one Y⁶ is a nitrogenatom. In many embodiments of the invention each Y⁶ where present is acarbon atom.

When an arylene group is present in the X² moiety this may besubstituted as indicated at any of the four positions (not connectingthe arylene group to the Effector and trigger termini of the compoundsof formula (I) that is) by substituents Z⁸ which may be selectedindependently as defined in claim 1.

Where X² comprises one or more amide or thioamide moieties—CH(Z⁹)NH thisis typically, where present, (each) Z⁹ is oxygen whereby to provide oneor more amide moieties although, where more than one Z⁹ is present, eachZ⁹ may be selected independently.

Finally, the Effector part of the compounds of formula (I) is the moietywhich provides the desired targeted effect in cells, typically those inwhich CYP1B1 is expressed. The Effector component may be any moleculehaving a pharmacological diagnostic or screening function when releasedfrom the compound of formula (I). By pharmacological or diagnosticfunction is meant that the effector component, when released, has adiscernable pharmacological or diagnostic effect on the cells in whichit is released.

It will be understood by those skilled in the art that the Effectorcomponent (Effector) in the compounds of formula (I) when released maycomprise an atom described herein as part of X¹—e.g. as oxygen or sulfuratom, or part of X², e.g. X³, e.g. an oxygen or sulfur atom. However, itis to be understood that the distinctions between the trigger, linkerand Effector portions of the compounds of formula (I) are made simply toassist in the description of the compounds of the invention; the skilledperson will be aware that the Effector portion in the compounds of theinvention constitutes the bulk of the Effector molecule that is releasedupon hyroxylation-induced breakdown but that one or some of the atoms inthe Effector molecule that is released may be provided by atomsdescribed herein as being X¹, part of X¹ or X² and indeed elsewhere(e.g. hydrogen atoms picked up from water molecules). Alternatively theEffector molecule may be attached to the remainder of the compounds offormula (I) through keto or fomyl groups for example.

The Effector molecule, where this has a pharmacological effect, may be,for example, any chemical that has a cytostatic or cytotoxic effect uponthe cell that serves to effect its release is expressed (e.g.CYP1B1-expressing cells). As is known, a cytotoxic molecule is amolecule that is toxic to cells whereas a cytostatic agent is one thatsuppresses the growth and/or replication of cells.

In certain embodiments of the invention the Effector molecule is acytotoxic agent. Examples of cytotoxic agents that may be used includebut are not limited to alkylating agents, antimitotic agents,antifolates, antimetabolites, DNA-damaging agents and enzyme inhibitors(e.g tyrosine kinase inhibitors). Specific examples of possiblecytotoxic drug moieties include but are not limited tobis(haloethyl)phosphoroamidates, cyclophosphamides, gemcitabine,cytarabine, 5-fluorouracil, 6-mercaptopurine, camptothecin, topotecan,doxorubicin, daunorubicin duocarmycin, etoposide, duetoposide,combretastatin A-4, vinblastine, vincristine, AQ4N, hydroxyurea,maytansines, enediyenes, epothilones, taxanes, bleomycins,calicheamicins, colchicine, dacarbazine, dactinomycin, epirubicin,epirubicin derivatives, fludarabine, hydroxyureapentatostatin,methotraxate, mitomycin, mitoxantrone, carboplatin, cisplatin, taxels,6-thioguanine, vinca alkaloids, platinum coordination complexes,anthracenediones, substituted ureas, methyl hydrazine derivatives, andnitrogen mustards.

In certain embodiments of the invention, the Effector molecule is aphosphoramide mustard, that is to say a phosphoric acid derivative inwhich one or two, typically two, of the hydroxyl groups of phosphoricacid are exchanged for a nitrogen mustard, or an oxygen- orsulfur-containing analogue thereof, and optionally the P(═O) replacedwith P(═S). A nitrogen mustard herein is defined as a non-specificallyalkylating amine, structurally related to mustard gas(1,5-dichloro-3-thiapentane), in which the sulfur atom is replaced witha nitrogen atom and, optionally, one chlorethyl side chain is replacedby a hydrogen atom or alkyl group, or one or both terminal chlorosubstituents are replaced by a leaving group such as bromo, iodo ormesylate (—OSO₂CH₃). Examples of phosphoramide mustards include thecompounds known as phosphoramide mustard (PM) and isophosphoramidemustard (IPM):

Thus, it will be noted that the compound PM is an example, as well asthe name of the class, of compounds known as phosphoramide mustardssince it may be regarded as a derivative of phosphoric acid in which oneof the hydroxyl groups has been exchanged for a nitrogen mustard (theother hydroxyl group being exchanged for an amino group (NH₂)).

In those embodiments of the invention in which the Effector molecule isa phosphoramide mustard, in which one or two, typically two, of thehydroxyl groups of phosphoric acid derivative are exchanged for anoxygen- or sulfur-containing analogue of a of nitrogen mustard, by thisis meant analogues of phosphoramide mustards in which the nitrogenmustard is replaced with an analogue in which one chloroethyl arm isabsent and the nitrogen atom exchanged for a sulfur or an oxygen atom.

In a particular embodiment of the present invention the Effectormolecule is connected to the remainder of the compound through an oxygenor sulfur atom and -Effector is of formula (II):

(wherein:

-   -   Z¹ is oxygen or sulfur;    -   each X⁴ is independently oxygen, sulfur or NZ¹³ wherein each-Z¹³        is independently —(CH₂)₂—Z¹⁴, -alkyl or -hydrogen; and    -   each Z¹⁴ is independently chloro, bromo, iodo, or mesylate).

In certain embodiments of the invention, Z¹² is oxygen. In these andother specific embodiments, each X⁴ is the same. In these and otherspecific embodiments, each X⁴ is NZ¹³. In these and other specificembodiments, each Z¹ is hydrogen. In these and other specificembodiments of the invention, each Z¹⁴ is the same and/or is bromo orchloro. In particular embodiments of the invention, each Z¹⁴ present(which may be two, three or four Z¹⁴ moieties) is bromo.

Alternatively, the Effector molecule may be one that fulfils adiagnostic function, for example allowing identification, or a fullerunderstanding of the nature, of a tumor in which, for example, CYP1B1 isexpressed. An example of a class of Effector molecules that arediagnostic molecules are fluorophoric molecules. These may be useful inthe diagnosis of cancerous cells. Examples of fluorophoric compoundsinclude coumarins, resorufins, fluoresceins and rhodamines and it is infact through a number of experiments conducted on compounds of theinvention comprising coumarins as the Effector molecule that theviability of the present invention has been demonstrated (see theexamples section below).

It will thus be appreciated that the compounds of formula (I) in whichEffector fulfils a diagnostic function may be of use in methods ofdiagnosis and such methods constitute further aspects of the presentinvention. Therefore, the invention provides a compound of formula (I),or a pharmaceutically acceptable salt, ester, amide or solvate thereof,for use in a method of diagnosis of a proliferative condition, forexample pre-malignant or malignant cellular proliferation, a cancer, aleukaemia, psoriasis, a bone disease, a fibroproliferative disorder orartherosclerosis, for example a proliferative condition selected frombladder, brain, breast, colon, head and neck, kidney, lung, liver,ovarian, prostate and skin cancer, said method comprising administeringan amount of a compound, or pharmaceutically acceptable salt, ester,amide or solvate of formula (I) to a subject having or suspected ofhaving such a proliferative and monitoring for the distribution ofreleased Effector molecules in the subject whereby to allow a diagnosisto be made.

Alternatively, the Effector may be one that fulfils a screeningfunction, for example as part of a model prodrug library collection, inorder to identify trigger and linker combinations that fragment whenactivated by CYP1B1 and allelic variants thereof. An example of a classof Effector molecules are fluorophoric molecules. Examples offluorophoric compounds include the well-known coumarins, resorufins,fluoresceins, and rhodamines. It is in fact through a number ofexperiments conducted on compounds of the invention comprising coumarinsas the Effector molecule that the viability of the present invention hasbeen demonstrated (see Example 1 in the section below). It will thus beappreciated that the compounds of formula (I) in which the effectorfulfils a screening function may be of use in identifying trigger andlinker combinations for the design and synthesis of prodrugs activatedby CYP1B1 and such methods constitute further aspects of the presentinvention.

It can be thus appreciated that compounds of formula (I) in which aneffector fulfils a screening function as part of a model prodrug librarycollection can be used in combination with cytochrome P450 substrateprediction models to guide the design and synthesis of prodrugs withselectivity for the example CYP1B1, and allelic variants thereof such asCYP1B1*3. For the purpose of clarity, the combination of the modelprodrug library with the substrate prediction model links substratespecificity to prodrug activation and fragmentation by CYP1B1, which isa fundamental design principle. Futhermore, it can be thus appreciatedthat compounds of formula (I) in which the effector fulfils a screeningfunction can be used in combination with cytochrome P450 substrateprediction models to guide the design and synthesis of prodrugs whichare not activated by normal tissue cytochrome P450s exemplified byCYP1A1, CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. An example of asubstrate prediction model is the Gaussian Kernel weighted k-NNalgorithm based on Tanimoto similarity searches on, but not limited to,descriptors such as extended connectivity fingerprints. Cytochrome P450substrate prediction models for prodrug design can be built onbioactivity databases derived from cytochrome P450 HTS from structurallydiverse compound collections. It is in fact through a number ofexperiments conducted on compounds of the invention comprising coumarinsas the effector molecule used in combination with a CYP1B1 substrateprediction model that the viability of the present invention has beendemonstrated (see the Examples 1 and 2).

Alternatively, the Effector may be one that fulfils a screening functionas part of a model prodrug library collection in order to identifytrigger and linker combinations that fragment when activated by CYP1B1and/or other cytochrome P450s and allelic variants thereofover-expressed in cancer and other proliferative conditions. An exampleof a class of Effector molecules are fluorophoric molecules. Examples offluorophoric compounds include coumarins, resorufins, fluoresceins andrhodamines. Examples of cytochrome P450s other than CYP1B1 which areover-expressed in cancer include CYP2A/2B, CYP2F1, CYP2R1, CYP2S1,CYP2U1, CYP2W1, CYP3A5, CYP3A7, CYP4Z¹, CYP26A1, and CYP51.

It can be thus appreciated that compounds of formula (I) in which aneffector fulfils a screening function aspart of a model prodrug librarycollection can be used in combination with cytochrome P450 substrateprediction models to guide the design and synthesis of prodrugs withselectivity for CYP1B1 and/or other cytochrome P450s and allelicvariants thereof over-expressed in cancer and other proliferativeconditions. An example of a class of Effector molecules are fluorophoricmolecules. Examples of fluorophoric compounds include coumarins,resorufins, fluoresceins and rhodamines. Examples of cytochrome P450sother than CYP1B1 which are over-expressed in cancer include CYP2A/2B,CYP2F1, CYP2R1, CYP2S1, CYP2U1, CYP2W1, CYP3A5, CYP3A7, CYP4Z¹, CYP26A1,and CYP51. An example of a substrate prediction model is the GaussianKernel weighted k-NN algorithm based on Tanimoto similarity searches on,but not limited to, descriptors such as extended connectivityfingerprints. Cytochrome P450 substrate prediction models for prodrugdesign can be built on bioactivity databases derived from cytochromeP450 HTS from structurally diverse compound collections.

According to the aspects and embodiments of the present inventionwhereby the Effector fulfils a screening function, for example accordingto the seventh aspect of the invention, a set of compounds willtypically comprise a plurality of compounds, for example comprising atleast 10, for example at least 20 compounds. In certain embodiments, theset may comprise up to 100, 1000, 10,000 or even 100,000 compounds. Suchsets of compounds, i.e. pluralities of compounds according to the firstaspect of the invention wherein the Effector is a fluorophore, as wellas other pluralities of compounds in which the Effector is not solimited and/or the compounds may be pharmaceutically acceptable salts,esters, amides or solvates, constitute a still further aspect of thepresent invention.

According to embodiments of the seventh aspect of this invention, wherea compound releases the fluorophore in step (a) but not, or only to amuch lesser extent, in step (b), by this is meant that the P450 enzymetypically releases at least 10-fold, e.g. at least 20-fold, more of saidfluorophore in step (a) as compared to step (b).

Where screening, e.g. according to embodiments of the seventh aspect ofthis invention yields a hit, e.g. and typically a compound that releasesthe fluorophore in step (a) but not, or only to a much lesser extent, instep (b), the method of the seventh aspect of the invention optionallyincludes additional the steps of:

(d) modeling compounds identical in structure to those identified instep (c) except that the fluorophore is replaced with a molecule havinga pharmacologic function for binding to an active site of saidcytochrome P450 enzyme; and

(e) synthesizing compounds modeled in step (d) that are predicted to besubstrates for said cytochrome P450 enzyme.

Alternatively, these steps ((d) and (e)) may be practised independentlyto the mandatory steps of the seventh aspect of this invention (i.e.(a)-(c)) and so constitute a still further embodiment of the presentinvention.

Typically the cytochrome P450 enzyme is selected from the groupconsisting of CYP1B1, CYP2S1, CYP2W1, CYP4Z¹ and allelic variantsthereof, for example CYP1B1 and allelic variants thereof, e.g. CYP1B1.

An aspect of the present invention is the use of primary human tumourcell lines of early passage number <20 in vitro derived from resectedcancer specimens. The primary head and neck squamous cell carcinoma celllines UT-SCCs described in Examples 4 and 5 below constitutively expressCYP1B1 at the mRNA and protein level and can be transplantedsubcutaneously into immune-deficient mice, (for example nude or serverecombined immune deficient SCID mice) with high engraftment rates togenerate primary human tumour xenografts where the constitive expressionof cytochrome P450 protein expression matches that of the originatingtumour in the patient. These primary human tumour xenograft models, bymaintaining cytochrome P450 mRNA/protein expression similarly to theoriginating patient tumour can therefore be used to assess the efficacyof a compound of the invention, wherein the Effector moiety is an agenthaving pharmacologic activity, in treating cancer. Furthermore, in theclinical context these primary human tumour xenograft models can be usedto check if responses of a compound of the invention, wherein theEffector moiety is an agent having pharmacologic activity, arecorrelated with clinical responses and outcomes, indicating usefulnessfor personalized chemotherapy. The primary human tumour models can alsobe used to compare the efficacy of a compound of claim 1, wherein theEffector moiety is an agent having pharmacologic activity with standardchemotherapeutic regimens and therefore to identify the most effectiveregimens for compounds of claim 1 alone or in combination with otherchemotherapeutic agents.

Furthermore, as part of this invention it is possible to derive primaryhuman tumour xenografts by directly implanting tumour tissue takendirectly resected from patients and implanting subcutaneously into, forexample, nude, SCID and nonobese diabetic/servere combined immunedeficient (NOD/SCID) mice. It is possible to generate first generationprimary human tumour xenografts for a range of different cancers whichwill retain the histological and genetic characteristics of theoriginating tumor and as such will constitutively express CYP1B1mRNA/protein at a level similar to the originating tumour. These primaryhuman tumour xenograft models, by maintaining CYP1B1 mRNA/proteinexpression similarly to the originating patient tumour can therefore beused to assess the efficacy of a compound of the invention, wherein theEffector moiety is an agent having pharmacologic activity, in treatingcancer. Furthermore, in the clinical context these primary human tumourxenograft models can be used to check if responses of a compound ofclaim 1, wherein the Effector moiety is an agent having pharmacologicactivity are correlated with clinical responses and outcomes, indicatingusefulness for personalized chemotherapy. The primary human tumourmodels can also be used to compare the efficacy of a compound of theinvention, wherein the Effector moiety is an agent having pharmacologicactivity, with standard chemotherapeutic regimens and therefore toidentify the most effective regimens for compounds of the inventionalone or in combination with other chemotherapeutic agents.

Where according to the eighth aspect of this invention, the cancer isresultant from implantation of a cell from an early passage cell linederived from a tissue taken directly from a tumor or a cancer thatexpresses said cytochrome P450 enzyme at levels similar to those fromthe tumor or cancer from which it originates, levels may be consideredto be similar if they are within 10% to those from the tumor or cancerfrom which it originates, for example within 5%.

For use according to the present invention, the compounds or aphysiologically acceptable salt, solvate, ester or amide thereofdescribed herein may be presented as a pharmaceutical formulation,comprising the compound or physiologically acceptable salt, ester, amideor other physiologically functional derivative thereof, together withone or more pharmaceutically acceptable carriers therefor and optionallyother therapeutic and/or prophylactic ingredients. Any carrier(s) areacceptable in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof.

Examples of physiologically acceptable salts of the compounds accordingto the invention include acid addition salts formed with organiccarboxylic acids such as acetic, lactic, tartaric, maleic, citric,pyruvic, oxalic, fumaric, oxaloacetic, isethionic, lactobionic andsuccinic acids; organic sulfonic acids such as methanesulfonic,ethanesulfonic, benzenesulfonic and p-toluenesulfonic acids andinorganic acids such as hydrochloric, sulfuric, phosphoric and sulfamicacids.

The determination of physiologically acceptable esters or amides,particularly esters is well within the skills of those skilled in theart.

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of the compounds described herein, which may beused in the any one of the uses/methods described. The term solvate isused herein to refer to a complex of solute, such as a compound or saltof the compound, and a solvent. If the solvent is water, the solvate maybe termed a hydrate, for example a mono-hydrate, di-hydrate, tri-hydrateetc, depending on the number of water molecules present per molecule ofsubstrate.

It will be appreciated that the compounds of the present invention mayexist in various stereoisomeric forms and the compounds of the presentinvention as hereinbefore defined include all stereoisomeric forms andmixtures thereof, including enantiomers and racemic mixtures. Thepresent invention includes within its scope the use of any suchstereoisomeric form or mixture of stereoisomers, including theindividual enantiomers of the compounds of formulae (I) or (II) as wellas wholly or partially racemic mixtures of such enantiomers.

It will also be understood by those skilled in the art that anticancerprodrugs, such as those described herein, can be targeted towardsparticular tumours by attachment of a tumour-targetting moiety such astumour-targetting peptide, for example small peptides identified throughthe development of phage-displayed peptide libraries. Such peptides orother moieties may assist in the targeting of conjugates that comprisethem to a particular cancer, particularly a solid tumour. Accordingly,the provision of such conjugates, i.e. of a compound of the inventionconjugated to a tumour-targeting moiety, forms a further aspect of thisinvention as do compositions, uses and methods described herein thatcomprise or involve use of such conjugates.

The compounds of the present invention may be prepared using reagentsand techniques readily available in the art and/or exemplary methods asdescribed hereinafter. It has been found that compounds of the presentinvention exhibit cytotoxicity in cells expressing CYP1B1 enzyme, butare substantially non-toxic in normal cells that do not express CYP1B1.Compounds of the invention may also exhibit cytotoxicity in cellsexpressing CYP1A1 enzyme. In practice, therefore, the compounds of theinvention are non-toxic pro-drugs that are converted (typically byCYP1B1) into cytotoxic agents.

Suitably, the compounds of the invention have a cytotoxicity IC₅₀ valueas defined below or less than 10 μM, advantageously less than 5 μM, forexample less than 1.0 μM or 0.5 μM.

In some embodiments, the cytotoxicity of a compound of the invention maybe measured by incubating the compound at different serial dilutionswith cells engineered to express CYP1B1. Suitably, said cells may beChinese Hamster Ovary (CHO) cells, which may contain recombinant CYP1B1and cytochrome P-450 reductase (CPR). High levels of functional enzymewhen co-expressed with human P-450 reductase may be achieved usingdihydrofolate reductase (DHFR) gene amplification. Typically, theengineered cells may be incubated with the compound and, after asuitable period of time (e.g., 96 hours), further incubated (e.g., for1.5 hours) with a suitable assay reagent to provide an indication of thenumber of living cells in culture. A suitable assay reagent is MTS (seebelow) which is bioreduced by cells into a formazan product that issoluble in tissue culture medium. The absorbance of the formazan productcan be directly measured at 510 nm, and the quantitative formazanproduct as measured by the amount of absorbance at 490 nm or 510 nm isdirectly proportional to the number of living cells in culture. Detailedmethods for determining the IC₅₀ value of a compound according to theinvention are described in Example 3 below.

By way of comparison, the IC₅₀ values of the compounds of the inventionmay also be measured in cells (e.g., Chinese Hamster Ovary cells) thatdo not contain CYP1B1, for example wild type CHO cells. The compounds ofthe invention may suitably have a fold selectivity for CYP1B1 expressingcells of at least 200, where the “fold selectivity” is defined as thequotient of the IC₅₀ value of a given compound in non-CYP1 expressingcells and the IC₅₀ value of the same compound in CYP1B1 expressingcells.

In some embodiments, the cytotoxicity of a compound of the invention maybe also measured by incubating the compound at different serialdilutions with primary head and neck tumour cells derived from patientswith head and neck squamous cell carcinoma as described in Example 4.

In some embodiments, the in vivo efficacy of a compound of the inventionmay be measured by implanting primary head and neck squamous cellcarcinoma tumour cells which constitutively express CYP1B1subcutaneously into the flank of a nude mouse to generate primary humantumour xenograft models and measuring the effect of prodrug treatment ontumour growth as described in Example 5.

As such, the present invention also embraces the use of one or more ofthe compounds of the invention, including the aforementionedpharmaceutically acceptable esters, amides, salts, solvates andprodrugs, for use in the treatment of the human or animal body bytherapy, particularly the treatment or prophylaxis of proliferativeconditions such, for example, as proliferative disorders or diseases, inhumans and non-human animals, including proliferative conditions whichare in certain embodiments of the invention characterised by cells thatexpress CYP1B1. More particularly, the invention comprehends the use ofone or more of the compounds of the invention for the treatment ofcancers characterised in certain embodiments of the invention by CYP1B1expression.

By “proliferative condition” herein is meant a disease or disorder thatis characterised by an unwanted or uncontrolled cellular proliferationof excessive or abnormal cells which is undesired, such as, neoplasticor hyperplastic growth, whether in vitro or in vivo. Examples ofproliferative conditions are pre-malignant and malignant cellularproliferation, including malignant neoplasms and tumours, cancers,leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g.,of connective tissues) and atherosclerosis.

Said proliferative condition may be characterised in certain embodimentsof the invention by cells that express CYP1B1.

Said proliferative condition may be selected from bladder, brain,breast, colon, head and neck, kidney, lung, liver, ovarian, prostate andskin cancer. In some embodiments, said proliferative condition maycomprise a solid tumour.

By “treatment” herein is meant the treatment by therapy, whether of ahuman or a non-human animal (e.g., in veterinary applications), in whichsome desired therapeutic effect on the proliferative condition isachieved; for example, the inhibition of the progress of the disorder,including a reduction in the rate of progress, a halt in the rate ofprogress, amelioration of the disorder or cure of the condition.Treatment as a prophylactic measure is also included. References hereinto prevention or prophylaxis herein do not indicate or require completeprevention of a condition; its manifestation may instead be reduced ordelayed via prophylaxis or prevention according to the presentinvention. By a “therapeutically-effective amount” herein is meant anamount of the one or more compounds of the invention or a pharmaceuticalformulation comprising such one or more compounds, which is effectivefor producing such a therapeutic effect, commensurate with a reasonablebenefit/risk ratio.

The compounds of the present invention may therefore be used asanticancer agents. By the term “anticancer agent” herein is meant acompound that treats a cancer (i.e., a compound that is useful in thetreatment of a cancer). The anti-cancer effect of the compounds of theinvention may arise through one or more mechanisms, including theregulation of cell proliferation, the inhibition of angiogenesis, theinhibition of metastasis, the inhibition of invasion or the promotion ofapoptosis.

It will be appreciated that appropriate dosages of the compounds of theinvention may vary from patient to patient. Determining the optimaldosage will generally involve the balancing of the level of therapeuticbenefit against any risk or deleterious side effects of the treatmentsof the present invention. The selected dosage level will depend on avariety of factors including the activity of the particular compound,the route of administration, the time of administration, the rate ofexcretion of the compound, the duration of the treatment, other drugs,compounds or materials used in combination and the age, sex, weight,condition, general health and prior medical history of the patient. Theamount of compound(s) and route of administration will ultimately be atthe discretion of the physician, although generally the dosage will beto achieve local concentrations at the site of action so as to achievethe desired effect.

Administration in vivo can be effected in one dose, continuously orintermittently throughout the course of treatment. Methods ofdetermining the most effective means and dosage of administration arewell known to a person skilled in the art and will vary with theformulation used for therapy, the purpose of the therapy, the targetcell being treated, and the subject being treated. Single or multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physician.

Pharmaceutical formulations include those suitable for oral, topical(including dermal, buccal and sublingual), rectal or parenteral(including subcutaneous, intradermal, intramuscular and intravenous),nasal and pulmonary administration e.g., by inhalation. The formulationmay, where appropriate, be conveniently presented in discrete dosageunits and may be prepared by any of the methods well known in the art ofpharmacy. Methods typically include the step of bringing intoassociation an active compound with liquid carriers or finely dividedsolid carriers or both and then, if necessary, shaping the product intothe desired formulation.

Pharmaceutical formulations suitable for oral administration wherein thecarrier is a solid are most preferably presented as unit doseformulations such as boluses, capsules or tablets each containing apredetermined amount of active compound. A tablet may be made bycompression or moulding, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing in asuitable machine an active compound in a free-flowing form such as apowder or granules optionally mixed with a binder, lubricant, inertdiluent, lubricating agent, surface-active agent or dispersing agent.Moulded tablets may be made by moulding an active compound with an inertliquid diluent. Tablets may be optionally coated and, if uncoated, mayoptionally be scored. Capsules may be prepared by filling an activecompound, either alone or in admixture with one or more accessoryingredients, into the capsule shells and then sealing them in the usualmanner. Cachets are analogous to capsules wherein an active compoundtogether with any accessory ingredient(s) is sealed in a rice paperenvelope. An active compound may also be formulated as dispersiblegranules, which may for example be suspended in water beforeadministration, or sprinkled on food. The granules may be packaged,e.g., in a sachet.

Formulations suitable for oral administration wherein the carrier is aliquid may be presented as a solution or a suspension in an aqueous ornon-aqueous liquid, or as an oil-in-water liquid emulsion.

Formulations for oral administration include controlled release dosageforms, e.g., tablets wherein an active compound is formulated in anappropriate release-controlling matrix, or is coated with a suitablerelease-controlling film. Such formulations may be particularlyconvenient for prophylactic use.

Pharmaceutical formulations suitable for rectal administration whereinthe carrier is a solid are most preferably presented as unit dosesuppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art. The suppositories may beconveniently formed by admixture of an active compound with the softenedor melted carrier(s) followed by chilling and shaping in moulds.

Pharmaceutical formulations suitable for parenteral administrationinclude sterile solutions or suspensions of an active compound inaqueous or oleaginous vehicles.

Injectable preparations may be adapted for bolus injection or continuousinfusion. Such preparations are conveniently presented in unit dose ormulti-dose containers, which are sealed after introduction of theformulation until required for use. Alternatively, an active compoundmay be in powder form that is constituted with a suitable vehicle, suchas sterile, pyrogen-free water, before use.

An active compound may also be formulated as long-acting depotpreparations, which may be administered by intramuscular injection or byimplantation, e.g., subcutaneously or intramuscularly. Depotpreparations may include, for example, suitable polymeric or hydrophobicmaterials, or ion-exchange resins. Such long-acting formulations areparticularly convenient for prophylactic use.

Formulations suitable for pulmonary administration via the buccal cavityare presented such that particles containing an active compound anddesirably having a diameter in the range of 0.5 to 7 microns aredelivered in the bronchial tree of the recipient.

As one possibility such formulations are in the form of finelycomminuted powders which may conveniently be presented either in apierceable capsule, suitably of, for example, gelatin, for use in aninhalation device, or alternatively as a self-propelling formulationcomprising an active compound, a suitable liquid or gaseous propellantand optionally other ingredients such as a surfactant and/or a soliddiluent. Suitable liquid propellants include propane and thechlorofluorocarbons, and suitable gaseous propellants include carbondioxide. Self-propelling formulations may also be employed wherein anactive compound is dispensed in the form of droplets of solution orsuspension.

Such self-propelling formulations are analogous to those known in theart and may be prepared by established procedures. Suitably they arepresented in a container provided with either a manually-operable orautomatically functioning valve having the desired spraycharacteristics; advantageously the valve is of a metered typedelivering a fixed volume, for example, 25 to 100 microlitres, upon eachoperation thereof.

As a further possibility an active compound may be in the form of asolution or suspension for use in an atomizer or nebuliser whereby anaccelerated airstream or ultrasonic agitation is employed to produce afine droplet mist for inhalation.

Formulations suitable for nasal administration include preparationsgenerally similar to those described above for pulmonary administration.When dispensed such formulations should desirably have a particlediameter in the range 10 to 200 microns to enable retention in the nasalcavity; this may be achieved by, as appropriate, use of a powder of asuitable particle size or choice of an appropriate valve. Other suitableformulations include coarse powders having a particle diameter in therange 20 to 500 microns, for administration by rapid inhalation throughthe nasal passage from a container held close up to the nose, and nasaldrops comprising 0.2 to 5% w/v of an active compound in aqueous or oilysolution or suspension.

It should be understood that in addition to the aforementioned carrieringredients the pharmaceutical formulations described above may include,an appropriate one or more additional carrier ingredients such asdiluents, buffers, flavouring agents, binders, surface active agents,thickeners, lubricants, preservatives (including anti-oxidants) and thelike, and substances included for the purpose of rendering theformulation isotonic with the blood of the intended recipient.

Pharmaceutically acceptable carriers are well known to those skilled inthe art and include, but are not limited to, 0.1 M and preferably 0.05 Mphosphate buffer or 0.8% saline. Additionally, pharmaceuticallyacceptable carriers may be aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's or fixed oils. Preservatives and other additives mayalso be present, such as, for example, antimicrobials, antioxidants,chelating agents, inert gases and the like.

Formulations suitable for topical formulation may be provided forexample as gels, creams or ointments.

Liquid or powder formulations may also be provided which can be sprayedor sprinkled directly onto the site to be treated, e.g. a wound orulcer. Alternatively, a carrier such as a bandage, gauze, mesh or thelike can be sprayed or sprinkle with the formulation and then applied tothe site to be treated.

Therapeutic formulations for veterinary use may conveniently be ineither powder or liquid concentrate form. In accordance with standardveterinary formulation practice, conventional water-soluble excipients,such as lactose or sucrose, may be incorporated in the powders toimprove their physical properties. Thus particularly suitable powders ofthis invention comprise 50 to 100% w/w and preferably 60 to 80% w/w ofthe active ingredient(s) and 0 to 50% w/w and preferably 20 to 40% w/wof conventional veterinary excipients. These powders may either be addedto animal feedstuffs, for example by way of an intermediate premix, ordiluted in animal drinking water.

Liquid concentrates of this invention suitably contain the compound or aderivative or salt thereof and may optionally include a veterinarilyacceptable water-miscible solvent, for example polyethylene glycol,propylene glycol, glycerol, glycerol formal or such a solvent mixed withup to 30% v/v of ethanol. The liquid concentrates may be administered tothe drinking water of animals.

In general, a suitable dose of the one or more compounds of theinvention may be in the range of about 1 μg to about 5000 μg/kg bodyweight of the subject per day, e.g., 1, 5, 10, 25, 50, 100, 250, 1000,2500 or 5000 μg/kg per day. Where the compound(s) is a salt, solvate,prodrug or the like, the amount administered may be calculated on thebasis the parent compound and so the actual weight to be used may beincreased proportionately.

In some embodiments, the one or more compounds of the present inventionmay be used in combination therapies for the treatment of proliferativeconditions of the kind described above, i.e., in conjunction with othertherapeutic agents. Examples of such other therapeutic agents includebut are not limited to topoisomerase inhibitors, alkylating agents,antimetabolites, DNA binders and microtubule inhibitors (tubulin targetagents), such as cisplatin, cyclophosphamide, doxorubicin, etoposide,irinotecan, fludarabine, 5FU, taxanes or mitomycin C. Other therapeuticagents will be evident to those skilled in the art. For the case ofactive compounds combined with other therapies the two or moretreatments may be given in individually varying dose schedules and viadifferent routes.

The combination of the agents listed above with a compound of thepresent invention would be at the discretion of the physician who wouldselect dosages using his common general knowledge and dosing regimensknown to a skilled practitioner.

Where a compound of the invention is administered in combination therapywith one, two, three, four or more, preferably one or two, preferablyone other therapeutic agents, the compounds can be administeredsimultaneously or sequentially. When administered sequentially, they canbe administered at closely spaced intervals (for example over a periodof 5-10 minutes) or at longer intervals (for example 1, 2, 3, 4 or morehours apart, or even longer period apart where required), the precisedosage regimen being commensurate with the properties of the therapeuticagent(s).

The compounds of the invention may also be administered in conjunctionwith non-chemotherapeutic treatments such as radiotherapy, photodynamictherapy, gene therapy, surgery and controlled diets.

The invention is now illustrated with reference to the followingnon-limiting examples:

Preparation of Compounds General

¹H, ¹³C and ³¹P nuclear magnetic resonance (NMR) spectra were recordedin the indicated solvent on either a Bruker Avance DPX 500 MHz or BrukerAvance 300 MHz spectrometer. Chemical shifts are expressed in ppm.Signal splitting patterns are described as singlet (s), broad singlet(bs), doublet (d), triplet (t), quartet (q), multiplet (m) orcombination thereof. Low resolution electrospray (ES) mass spectra wererecorded on a Bruker MicroTof mass spectrometer, run in a positive ionmode, using either methanol/water (95:5) or water acetonitrile(1:1)+0.1% formic acid as a mobile phase. High resolution electrospraymeasurements were performed on a Bruker Microtof mass spectrometer.LC-MS analysis were performed with an Agilent HPLC 1100 (PhenomenexGemini Column 5μ C18 110 Å 50×3.0 mm, eluted with (0 to 20% MeOH/H₂) anda diode array detector in series with a Bruker Microtof massspectrometer. Column chromatography was performed with silica gel(230-400 mesh) or RediSep®4, 12, 40 or 80 g silica prepacked columns.All the starting materials are commercially available and were usedwithout further purification. All reactions were carried out under dryand inert conditions unless otherwise stated.

[Compounds indicated below with a parenthetical dagger (†) are notexamples of the invention but are included for a better understanding ofit.]

1. Phosphoroamidate Mustard Prodrugs

Code Structure SU025-04

SU046-04

Synthesis of the Phosphoroamidate Prodrugs SU025-04 and SU046-04

1. Synthesis of the trigger component of the prodrugs

2-Bromo-3,5-dimethoxybenzaldehyde (1)

3,5-dimethoxybenzaldehyde (12.6 g, 76 mmol) was dissolved in acetic acid(350 mL). The resulting colourless solution was cooled to 0° C. Asolution of bromine (3.9 mL) in ethanoic acid (50 mL) was added dropwiseover 1 h. Once the addition was complete the ice bath was removed andthe resulting pale green solution was stirred overnight at roomtemperature. Cold water was added to the solution. The resulting whitesolid was collected by vacuum filteration and rinsed with water. Thesolid was then redissolved in EtOAc and adsorbed on silica gel. Theproduct was purified by flash chromatography, eluting with hexane/EtOAc(4:1) to give 1 (12.5 g, 66%) as a white solid. m/z=345.98 (M+H). ¹H NMR(500 MHz, CDCl₃): δ: 10.43 (1H, s, CHO), 7.06 (1H, s, ArH), 6.73 (1H, s,ArH), 3.93 (3H, s, CH₃O), 3.86 (3H, s, CH₃O). ¹³C NMR (500 MHz, CDCl₃):δ: 192.09 (CHO), 159.92 (C-5), 157.02 (C-3), 134.67 (C-1), 109.12 (C-2),105.83, 103.37 (C-4 & C-6), 56.60 (OMe), 55.82 (OMe).

2-Hydroxy-3,5-dimethoxybenzaldehyde (2)

Morpholine (2.05 g, 24 mmol) and THF (40 mL) were placed in athree-necked, round bottomed flask equipped with a stirring bar, septumcap, dropping funnel, thermometer, and argon inlet. The flask was cooledin a dry ice-acetone bath to −50° C., and a solution of n-BuLi in hexane(1.6M, 15 mL, 24 mmol) was added all at once. After 10 min a solution of1 (4.9 g, 20 mmol) in THF (30 mL) was added dropwise via a syringe overa period of 4 min, and the mixture was cooled to ˜−75° C. over 20 min.n-BuLi in hexane (1.6M, 20 mL, 32 mmol) was then added dropwise over 45min, keeping the temperature at −75° C. After complete addition ofn-BuLi the solution was stirred for 35 min. A solution of nitrophenol(6.90 g, 46 mmol) in 10 mL THF was added from the dropping funnel,keeping the temperature at −75° C. The resulting dark mixture wasstirred at −75° C. for 4 h and then allowed to warm to room temperature.It was acidified to pH 1 with 6N HCl and stirred for 15 min. Afterdilution with brine (100 mL), THE was removed in vacuo. The aqueoussolution was extracted with diethyl ether (4×40 mL). The combinedorganic layers were extracted with 2 N NaOH (3×40 mL). The combined NaOHextracts were washed with diethyl ether (3×20 mL) and then acidified topH 1 with concentrated HCl. The resulting mixture was extracted withCH₂Cl₂ (3×20 mL), and the combined organic extracts were washed withbrine, dried (MgSO₄) and adsorbed on silica gel. The product waspurified by flash chromatography, eluting with EtOAc/hexane (1:2). Pure2 was obtained (2.0 g, 55%) as a yellow solid. m/z=183.06 (M+H). ¹H NMR(500 MHz, CDCl₃): δ: 10.71 (1H, s, OH), 9.91 (1H, s, CHO), 6.77 (1H, d,J_(4, 6)=2.8 Hz, H-6), 6.61 (1H, d, J_(4, 6)=2.8 Hz, H-4), 3.92 (3H, s,OMe), 3.84 (3H, s, OMe). ¹³C NMR CDEPT135 (500 MHz, CDCl₃): δ: 196.11(CHO), 107.93 (C-6), 103.90 (C-4), 56.29 (OMe), 55.83 (OMe).

2-(2,2-Diethoxyethoxy)-3,5-dimethoxybenzaldehyde (3)

To a stirred suspension containing 2 (1.1 g, 6.0 mmol) and K₂CO₃ (1.0 g,7.2 mmol) in DMF (100 mL), bromoacetaldehyde diethyl acetal (0.93 mL,6.0 mmol) was added dropwise. The mixture was refluxed for 4 h. Aftercooling, the precipitate was filtered off and the solvent was evaporatedin vacuo. The crude residue was adsorbed on silica gel and purified byflash chromatography, eluting with hexane/EtOAc (4:1) to give 3 (1.2 g,67%) as a clear oil. ¹H NMR (500 MHz, CDCl₃): δ: 10.50 (1H, s, CHO),6.88 (1H, d, J_(4, 6)=2.9 Hz, H-6), 6.74 (1H, d, J_(4, 6)=2.9 Hz, H-4),4.83 (1H, t, J_(4, 6)=5.3 Hz, CH), 4.14 (2H, d, J_(4, 6)=5.3 Hz, CH₂),3.88 (3H, s, OMe), 3.83 (3H, s, OMe), 3.77-3.71) (2H, m, CH₂CH₃),3.63-3.58 (2H, m, CH₂CH₃), 1.24 (6H, t, J=7.1 Hz, 2×CH₃).

5,7-dimethoxybenzofuran-2-carbaldehyde (4)

A stirred solution of 3 (1.2 g, 4.0 mmol) in acetic acid (35 mL) wasrefluxed for 16 h. After cooling, the solution was evaporated todryness. The crude product was adsorbed on silica gel and purified byflash chromatography, eluting with hexane/EtOAc (2:1) to give 4 (230 mg,28%) as a white solid. ¹H NMR (500 MHz, CDCl₃): δ: 9.89 (1H, s, CHO),7.50 (1H, s, H-3), 6.69 (1H, d, J_(4, 6)=2.2 Hz, H-6), 6.64 (1H, d,J_(4, 6)=2.2 Hz, H-4), 4.01 (3H, s, OMe), 3.87 (3H, s, OMe). ¹³C NMRCDEPT 135, (500 MHz, CDCl₃): δ: 179.91 (CHO), 153.37 (C-2), 116.10(C-3), 101.80 (C-6), 94.90 (C-4), 56.20 (OMe), 55.88 (OMe).

(5,7-dimethoxybenzofuran-2-yl)methanol (5)

Compound 4 (460 mg, 2.23 mmol) was dissolved in THF (5 mL) and EtOH (1mL). NaBH₄ (102 mg, 2.68 mmol) was added portionwise at 0° C., withvigorous stirring. The suspension was stirred at 0° C. for 15 min andthen at room temperature for 1 h. Solvents were evaporated off in-vacuo.The crude residue was taken up in EtOAc and washed with water, brine anddried (MgSO₄). The residue was adsorbed on silica gel and purified byflash chromatography, eluting with hexane/EtOAc (1:1) to give 5 (388 mg,82%) as an oil. m/z=209.08 (M+H). ¹H NMR (500 MHz, CDCl₃): δ: 6.62 (1H,s, H-3), 6.60 (1H, s, H-6), 6.46 (1H, s, H-4), 4.76 (2H, s, 2-CH₂), 3.99(3H, s, OMe), 3.85 (3H, s, OMe). ¹³C NMR (500 MHz, CDCl₃): δ: 157.23(C-5), 156.72 (C-1), 145.36 (C-7), 139.50 (C-1a), 129.38 (C-4a), 104.62(C-3), 96.96 (C-6), 94.58 (C-4), 57.98 (2-CH₂), 55.95 (OMe), 55.83(OMe).

2. Synthesis of the Effector Components of the Prodrugs

N,N-bis (2-chloroethyl)phosphonamidic Acid (6)

To a suspension of 2-chloroethylamine hydrochloride (7.2 g, 62 mmol) inCH₂Cl₂ (110 mL) was added POCl₃ (2.84 mL, 31 mmol) over 15 min at −78°C. with vigorous stirring, followed by the addition of a solution of TEA(17.5 mL, 124 mmol) in CH₂Cl₂ (30 mL) over 4 h. The reaction mixture wasstirred at −78° C. for 1 h and then allowed to warm to room temperatureand stirred for 2 h. The resulting solid was filtered and washed withcold EtOAc. The solid was discarded. The filtrate was concentrated undervacuum to about 5 mL and EtOAc was added (10 mL). The resultingsuspension was filtered and washed with EtOAc (2×10 mL). The solid wasagain discarded. The filtrate was concentrated under vacuum to dryness.The residue was then dissolved in THF (7 mL) followed by addition of anaqueous NaBr solution (NaBr (5 g) in 100 mL water) at 0° C. over 20 min.The mixture was then warmed to room temperature and stirred for 15 h ina water bath. A white solid precipitated from the reaction mixture. Themixture was then kept at −20° C. in the freezer for 2 h. The crystallinesolid was filtered and washed with cold water (2×50 mL, 0° C.) and coldEtOAc (2×50 mL, 0° C.). After drying at room temperature under vacuumovernight, the product 6 was obtained (1.8 g, 26%) as a white solid. ¹HNMR (500 MHz, DMSO-d₆): δ: 5.29 (3H, br, OH & NH), 3.55 (4H, t, J=7.0Hz, 2×CH₂), 3.01 (4H, dt, J=12.2, 7.0 Hz, 2×CH₂). ³¹P NMR (500 MHz,DMSO-d₆) δ: 12.28 ppm.

N,N-bis (2-bromoethyl)phosphonamidic Acid (7)

Compound 7 was synthesized using a similar method as above. It wasobtained in 18% yield (1.64 g). ¹H NMR (500 MHz, DMSO-d₆): δ: 6.08 (3H,s, OH & NH), 3.46 (4H, t, J=7.0 Hz, 2×CH₂), 3.01 (4H, dt, J=12.2, 7.0Hz, 2×CH₂). ³¹P NMR (500 MHz, DMSO-d₆) δ: 12.23 ppm.

3. Coupling Reaction for the Synthesis of SU025-04 and SU046-04

5,7-Dimethoxybenzofuran-2-yl)methylN,N′-bis(2-chloroethyl)phosphordiamidate(8) SU025-04

To a suspension of 5 (300 mg, 1.44 mmol), 6 (479 mg, 2.16 mmol) and PPh₃(565 mg, 2.16 mmol) in THF (20 mL) was added DIAD (0.426 mL, 2.16 mmol),dropwise at 0° C. The resulting suspension was warmed to roomtemperature and stirred for 2 h. The solvent was removed, and theresidue was purified by flash chromatography (70% acetone in toluene) togive 8 (250 mg, 42%) as an oil. m/z=823.08 (2M+H). ¹H NMR (500 MHz,DMSO-d₆): δ 7.27 (2H, s, 2×NH), 6.75 (1H, s, ArH-3), 6.62 (1H, d,J_(4, 6)=2.3 Hz, ArH-4), 6.49 (1H, d, J_(4, 6)=2.3 Hz, ArH-6), 5.13 (2H,d, J=9.3 Hz, 2H), 3.99 (3H, s, OMe), 3.86 (3H, s, OMe), 3.29 (4H, m,2×CH₂), ³¹P NMR (500 MHz, DMSO-d₆) δ:14.76 ppm. HRMS: Calcd forC₁₅H₂₁N₂O₅PCl₂Na, 433.0463; found 433.0471.

5,7-Dimethoxybenzofuran-2-yl)methylN,N′-bis(2-bromoethyl)phosphordiamidate(9) SU046-04

Compound 9 (SU046-04) was synthesized using a similar method as above.It was obtained in 25% yield (10 mg). m/z=500.96 (M+H), 1000.93 (2M+H).¹H NMR (500 MHz, CDCl₃): δ 6.76 (1H, s, ArH-3), 6.61 (1H, d,J_(4, 6)=2.2 Hz, ArH-4), 6.48 (1H, d, J_(4, 6)=2.2 Hz, ArH-6), 5.13 (2H,d, J=9.4 Hz, 2H), 3.99 (3H, s, OMe), 3.86 (3H, s, OMe), 3.49-3.45 (4H,m, 2×CH₂), 3.40-3.33 (4H, m, 2×CH₂) 3.23 (2H, bs, NH). ¹³C NMR (500 MHz,CDCl₃): δ: 156.98, 152.91, 145.58, 139.97, 129.06, 128.24, 107.45,97.70, 94.56, 59.73, 56.00, 42.90, 34.76, 30.98. HRMS: Calcd forC₁₅H₂₁N₂O₅PBr₂Na, 520.9453; found 520.9454.

2. Ether and Thioether-Linked Model Prodrugs

TLE- M2- SU010A

VG015- 05

VG016- 05 (†)

VG017- 05

VG027- 05

VG029- 05

VG035- 04

VG028- 05

VG035- 05

TLE- M1- SU001A (†)

VG040- 03

TLE- M1- SU004A (†)

VG039- 03

SU06- 02 (†)

SU010- 02 (†)

VG033- 03

VG015- 04 (†)

VG014- 04

VG015-02 (†)

Synthesis of Ether and Thioether Linked Prodrugs7-(benzofuran-2-ylmethoxy)-4-methyl-2H-chromen-2-one (10) TLE-M2-SU010A

2-(bromomethyl)benzofuran (10)

Benzofuran-2-yl methanol (1.0 g, 6.7 mmol) was dissolved in toluene (50mL) and pyridine (653 μL, 8.1 mmol) was added. The solution was cooledto 0° C. PBr₃ (760 μL, 8.1 mmol) was added dropwise over 15 min. Thereaction mixture was then brought up to room temperature and stirred for1 h. The mixture was washed with K₂CO 3 solution and extracted withEtOAc (3×30 mL). The EtOAc layer was washed with brine and dried(MgSO₄). The solvent was evaporated off in-vacuo and product waspurified by flash chromatography, eluting with hexane:EtOAc (4:1) togive 10 (780 mg, 55%) as an oil. ¹H NMR (500 MHz, CDCl₃): δ: 7.57 (1H,d, J=7.75 Hz, H-4), 7.52 (1H, d, J=8.40, H-7), 7.35 (1H, t, J=8.90,H-4), 7.27 (1H, t, J=8.9 Hz, H-5), 6.79 (1H, s, H-3), 4.64 (2H, s,2-CH₂). ¹³C NMR CDEPT 135, (500 MHz, CDCl₃): δ: 155.34 (C-2), 152.65(C-7a), 129.08 (C-3a), 125.20 (C-6), 123.16 (C-5), 121.34 (C—C-4),111.46 (C-7), 106.30 (C-3), 23.61 (CH₂—Br).

7-(benzofuran-2-ylmethoxy)-4-methyl-2H-chromen-2-one (11) TLE-M2-SU010A

Sodium ethoxide (77 mg, 1.13 mmol) was added to DMF (10 mL) at 0° C.,and the suspension was stirred for 10 min. 7-hydroxy-4-methylcoumarin(200 mg, 1.13 mmol) was slowly added and resulting mixture was stirredat this temp for 0.5 h. To this mixture 10 (200 mg, 0.94 mmol) was addedportionwise. Resulting reaction mixture was stirred at room temperaturefor 2 h. DMF was evaporated off in-vacuo and the residue was taken up inEtOAc, and washed with brine, water and 1M NaOH (2×30 mL). The organiclayer was dried (MgSO₄) and product was purified by flashchromatography, eluting with hexane: EtOAc (2:1) to give 11 (35 mg, 12%)as a white solid. m/z=307 (M+H). H¹ NMR (500 MHz, DMSO-d₆): δ=7.75-7.60(2H, m, ArH), 7.40-7.25 (2H, m, ArH), 7.23 (1H, s, ArH), 7.12 (2H, d,CH), 6.25 (1H, s, CH), 5.41 (2H, s, CH₂), 2.38 (3H, s, CH₃).

7-((5-fluorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (16)VG015-05

2-(2,2-Diethoxyethoxy)-5-fluorobenzaldehyde (12)

To a stirred suspension containing 2-hydroxy-5-fluorobenzaldehyde (500mg, 3.57 mmol) and K₂CO₃ (524 mg, 13.79 mmol) in DMF (10 mL),bromoacetaldehyde diethyl acetal (0.6 mL, 3.93 mmol) was added dropwise.The mixture was refluxed for 4 h. After cooling, the precipitate wasfiltered off and the solvent was evaporated in vacuo. The crude residuewas adsorbed on silica gel and purified by flash chromatography, elutingwith hexane/EtOAc (4:1) to give 12 (300 mg, 33%) as an oil. ¹H NMR (500MHz, CDCl₃): δ: 10.30 (1H, s, CHO), 7.31 (1H, d, J=7.85 Hz), 7.10 (1H,t, J=7.80 Hz), 6.87 (1H, d, J=8.86 Hz), 4.75 (1H, s, CH), 3.97 (2H, d,J=2.35 Hz, CH₂), 3.67-3.64 (2H, m, CH₂CH₃), 3.53-3.50 (2H, m, CH₂CH₃),1.11 (6H, t, J=6.00 Hz, 2×CH₃).

5-fluorobenzofuran-2-carbaldehyde (13)

A stirred solution of 12 (300 mg, 4.0 mmol) in acetic acid (10 mL) wasrefluxed for 24 h. After cooling, the solution was evaporated todryness. The crude product was adsorbed on silica gel and purified byflash chromatography, eluting with hexane/EtOAc (4:1) to give the 13(180 mg, 94%) as a white solid, ¹H NMR (500 MHz, CDCl₃): δ: 9.89 (1H, s,CHO), 7.57 (2H, m, ArH), 7.41 (1H, d, J=7.20 Hz), 7.26 (1H, d, J=6.94Hz, H-4). ¹³C NMR CDEPT 135, (500 MHz, CDCl₃): δ: 179.74 (CHO), 117.73(C-3), 117.25 (C-7), 113.81 (C-6), 108.68 (C-4).

(5-fluorobenzofuran-2-yl)methanol (14)

Compound 13 (180 mg, 1.10 mmol) was dissolved in EtOH (12 mL). NaBH₄ (45mg, 1.21 mmol) was added portionwise at 0° C., with vigorous stirring.The suspension was stirred at 0° C. for 15 min and then at roomtemperature for 1.5 h. Solvents were evaporated off in-vacuo. The cruderesidue was taken up in EtOAc and washed with water, brine and dried(MgSO₄). The solvent was evaporated off in vacuo. The residue wasadsorbed on silica gel and purified by flash chromatography, elutingwith hexane/EtOAc (3:1) to give 14 (150 mg, 91%) as a white solid. ¹HNMR (500 MHz, CDCl₃): δ: 7.36 (1H, d, J=8.35 Hz, H-7), 7.19 (1H, d,J=7.80, H-4), 7.00 (1H, t, J=8.75H-6), 6.60 (1H, s, H-3), 4.75 (2H, s,CH₂).

2-(bromomethyl)-5-fluorobenzofuran (15)

Compound 14 (150 mg, 0.90 mmol) was dissolved in toluene (10 mL) and thesolution was cooled to 0° C. PBr₃ (102 μL, 1.08 mmol) was added dropwiseover 15 min. The reaction mixture was then brought up to roomtemperature and stirred for 1 h. The solvent was evaporated offin-vacuo. The residue was adsorbed on silica gel and purified by flashchromatography, eluting with hexane/EtOAc (4:1) to give 15 (150 mg, 72%)as an oil. ¹H NMR (500 MHz, CDCl₃): δ: 7.43 (1H, d, J=7.60 Hz, H-7),7.21 (1H, t, J=8.10, H-4), 7.06 (1H, t, J=8.90, H-4), 6.75 (1H, s, H-3),4.60 (2H, s, 2-CH₂). ¹³C NMR CDEPT 135, (500 MHz, CDCl₃): δ: 113.08(C-7), 112.08 (C-6), 106.87 (C-4), 106.34 (C-3), 60.42 (CH₂—Br).

7-((5-fluorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (16)VG015-05

Sodium ethoxide (8.9 mg, 0.13 mmol) was added to DMF (3 ml) at 0° C.,and the suspension was stirred for 10 min. 7-hydroxy-4-methylcoumarin(25.4 mg, 0.14 mmol) was slowly added and resulting mixture was stirredat this temp for 0.5 h. To this mixture 15 (30 mg, 0.13 mmol) was addedportionwise. Resulting reaction mixture was stirred at room temperaturefor 2 h. DMF was evaporated off in-vacuo and the residue was purified byflash chromatography, eluting with hexane: EtOAc (3:1) to give 16 (9.0mg, 21%) as a white solid. m/z=325.20 (M+H).

7-((5,7-difluorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (19)VG016-05

2-(2,2-diethoxyethoxy)-3,5-difluorobenzaldehyde (17)

To a stirred suspension containing 2-hydroxy-3,5-fluorobenzaldehyde (1.0g, 6.32 mmol) and K₂CO₃ (960 mg, 6.95 mmol) in DMF (10 mL),bromoacetaldehyde diethyl acetal (1.07 mL, 6.95 mmol) was addeddropwise. The mixture was refluxed for 4 h. After cooling, theprecipitate was filtered off and the solvent was evaporated in vacuo.The crude residue was adsorbed on silica gel and purified by flashchromatography, eluting with hexane/EtOAc (4:1) to give 17 (380 mg, 22%)as an oil, ¹H NMR (500 MHz, CDCl₃): δ: 10.41 (1H, s, CHO), 7.29 (1H, d,J=6.80 Hz), 7.10 (1H, t, J=8.30 Hz), 4.80 (1H, s, CH), 4.20 (2H, d,J=3.25 Hz, CH₂), 3.71 (2H, t, J=7.10 Hz, CH₂CH₃), 3.57 (2H, t, J=7.45Hz, CH₂CH₃), 1.19 (6H, t, J=6.25 Hz, 2×CH₃).

2-(bromomethyl)-5,7-difluorobenzofuran (18)

A stirred solution of 17 (380 mg, 1.39 mmol) in acetic acid (10 mL) wasrefluxed for 24 h. After cooling, the solution was evaporated todryness. The crude product (300 mg) was dissolved in EtOH (5 mL). NaBH₄(73 mg, 1.98 mmol) was added portionwise at 0° C., with vigorousstirring. The suspension was stirred at 0° C. for 15 min and then atroom temperature for 1.5 h. Solvents were evaporated off in-vacuo. Thecrude residue (280 mg) was dissolved in toluene (20 mL) and the solutionwas cooled to 0° C. PBr₃ (142 μL, 1.52 mmol) was added dropwise over 15min. The reaction mixture was then brought up to room temperature andstirred for 1 h. The solvent was evaporated off in-vacuo. The residuewas adsorbed on silica gel and purified by flash chromatography, elutingwith hexane/EtOAc (4:1) to give 18 (210 mg, 61%). ¹H NMR (500 MHz,CDCl₃): δ: 7.03 (1H, d, J=7.50 Hz, H-4), 6.87 (1H, t, J=9.80, H-5), 6.79(1H, s, H-3), 4.59 (2H, s, 2-CH₂).

7-((5,7-difluorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (19)VG016-05

Sodium ethoxide (8.9 mg, 0.13 mmol) was added to DMF (3 mL) at 0° C.,and the suspension was stirred for 10 min. 7-hydroxy-4-methylcoumarin(25.4 mg, 0.14 mmol) was slowly added and resulting mixture was stirredat this temp for 0.5 h. To this mixture2-(bromomethyl)-5-fluorobenzofuran (30 mg, 0.12 mmol) was addedportionwise. Resulting reaction mixture was stirred at room temperaturefor 2 h. DMF was evaporated off in-vacuo and the residue was purified byflash chromatography, eluting with hexane: EtOAc (3:1) to give 19 (8.8mg, 21%) as a white solid. m/z=343.12 (M+H).

7-((5,7-difluorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one(22)(VG017-05)

2-(2,2-diethoxyethoxy)-5-fluoro-3-methylbenzaldehyde (20)

To a stirred suspension containing5-fluoro-2-hydroxy-3-methylbenzaldehyde (1.0 g, 6.49 mmol) and K₂CO₃(980 mg, 7.10 mmol) in DMF (8 mL), bromoacetaldehyde diethyl acetal(1.10 mL, 7.15 mmol) was added dropwise. The mixture was refluxed for 4h. After cooling, the precipitate was filtered off and the solvent wasevaporated in vacuo. The crude residue was adsorbed on silica gel andpurified by flash chromatography. The product was eluted withhexane/EtOAc (4:1) to give 20 (350 mg, 20%) as an oil, ¹H NMR (500 MHz,CDCl₃): δ: 10.40 (1H, s, CHO), 7.32 (1H, d, J=7.30 Hz, ArH), 7.14 (1H,d, J=7.75 Hz, ArH), 4.85 (1H, s, CH), 3.95 (2H, s, CH₂), 3.75 (2H, t,J=7.15 Hz, CH₂CH₃), 3.61 (2H, t, J=7.20 Hz, CH₂CH₃), 2.36 (3H, s, CH₃),1.24 (6H, t, J=5.65 Hz, 2×CH₃).

2-(bromomethyl)-5-fluoro-7-methylbenzofuran (21)

A stirred solution of 20 (350 mg, 1.30 mmol) in acetic acid (10 mL) wasrefluxed for 24 h. After cooling, the solution was evaporated todryness. The crude product (300 mg) was dissolved in THF (5 mL). NaBH₄(78 mg, 2.02 mmol) was added portionwise at 0° C., with vigorousstirring. The suspension was stirred at 0° C. for 15 min and then atroom temperature for 1.5 h. Solvents were evaporated off in-vacuo. Thecrude residue (260 mg) was dissolved in toluene (20 mL) and the solutionwas cooled to 0° C. PBr₃ (135 μL, 1.44 mmol) was added dropwise over 15min. The reaction mixture was then brought up to room temperature andstirred for 1 h. The solvent was evaporated off in-vacuo. The residuewas adsorbed on silica gel and purified by flash chromatography, elutingwith hexane/EtOAc (4:1) to give 21 (200 mg, 36%). ¹H NMR (500 MHz,CDCl₃): δ: 7.03 (1H, d, J=7.50 Hz, H-4), 6.88 (1H, t, J=9.80, H-5), 6.75(1H, s, H-3), 4.69 (2H, s, 2-CH₂), 2.54 (3H, s, CH₃).

7-((5,7-difluorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (22)VG017-05

Sodium ethoxide (8.9 mg, 0.13 mmol) was added to DMF (3 mL) at 0° C.,and the suspension was stirred for 10 min. 7-hydroxy-4-methylcoumarin(25.4 mg, 0.14 mmol) was slowly added and resulting mixture was stirredat this temp for 0.5 h. To this mixture 21 (30 mg, 0.12 mmol) was addedportionwise. Resulting reaction mixture was stirred at room temperaturefor 2 h. DMF was evaporated off in-vacuo and the residue was purified byflash chromatography, eluting with hexane: EtOAc (3:1) to give 22 as awhite solid (11 mg, 26%). m/z=33.20 (M+H).

7-((5-methoxybenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (27)VG027-05

2-(2,2-diethoxyethoxy)-5-methoxybenzaldehyde (23)

To a stirred suspension containing 2-hydroxy-5-methoxybenzaldehyde (2.0g, 13.16 mmol) and K₂CO₃ (2.18 g, 15.79 mmol) in DMF (20 mL),bromoacetaldehyde diethyl acetal (2.43 mL, 15.79 mmol) was addeddropwise. The mixture was refluxed for 4 h. After cooling, theprecipitate was filtered off and the solvent was evaporated in vacuo.The crude residue was adsorbed on silica gel and purified by flashchromatography. The product was eluted with hexane/EtOAc (4:1) to givethe target compound 23 (1.10 g, 31%) as an oil. ¹H NMR (500 MHz, CDCl₃):δ: 10.49 (1H, s, CHO), 7.33 (1H, d, J=3.30 Hz, ArH), 7.12 (1H, dd,J=5.75 & 3.30 Hz, ArH), 6.97 (1H, d, J=9.05 Hz), 4.87 (1H, t, J=5.25 Hz,CH), 4.09 (2H, d, J=5.25 Hz, CH₂), 3.81-3.78 (2H, m, CH₂CH₃), 3.67-3.64(2H, m, CH₂CH₃), 1.26 (6H, t, J=7.05 Hz, 2×CH₃).

5-methoxybenzofuran-2-carbaldehyde (24)

A stirred solution of 23 (1.0 g, 3.74 mmol) in acetic acid (10 mL) wasrefluxed for 16 h. After cooling, the solution was evaporated todryness. The crude product was adsorbed on silica gel and purified byflash chromatography, eluting with hexane/EtOAc (4:1) to give 24 (160mg, 24%) as a white solid. ¹H NMR (500 MHz, CDCl₃): δ: 9.80 (1H, s,CHO), 7.49-7.45 (2H, m, ArH), 7.12-7.09 (2H, m, ArH), 3.85 (3H, s, OCH).

(5-methoxybenzofuran-2-yl)methanol (25)

Compound 24 (3.5 g, 19.9 mmol) was dissolved in EtOH (20 mL). NaBH₄ (957mg, 25.87 mmol) was added portionwise at 0° C., with vigorous stirring.The suspension was stirred at 0° C. for 15 min and then at roomtemperature for 1.5 h. Solvent was evaporated off in-vacuo. The residuewas adsorbed on silica gel and purified by flash chromatography, elutingwith hexane/EtOAc (2:1) to give 25 (3.0 g, 85%) as a white solid. ¹H NMR(500 MHz, CDCl₃): δ: 7.36 (1H, d, J=8.90 Hz, H-7), 7.02 (1H, d, J=2.6,H-4), 6.90 (1H, dd, J=6.30 & 2.60, H-6), 6.61 (1H, s, H-3), 4.76 (2H, s,2-CH₂), 3.86 (3H, s, OCH₃), 2.16 (1H, bs, OH). ¹³C NMR CDEPT 135, (500MHz, CDCl₃): δ: 113.07 (C-7), 111.69 (C-6), 104.34 (C-4), 103.60 (C-3),58.24 (CH₂), 55.92 (OCH₃).

2-(bromomethyl)-5-methoxybenzofuran (26)

Compound 25 (40 mg, 0.22 mmol) was dissolved in toluene (5 mL) and thesolution was cooled to 0° C. PBr₃ (21 μL, 0.22 mmol) was added dropwiseover 10 min. The reaction mixture was then brought up to roomtemperature and stirred for 1 h. The solvent was evaporated offin-vacuo. The residue was adsorbed on silica gel and purified by flashchromatography, eluting with hexane/EtOAc (4:1) to give 26 (40 mg, 74%)as an oil. ¹H NMR (500 MHz, CDCl₃): δ: 7.39 (1H, d, J=8.90 Hz, H-7),7.01 (1H, d, J=2.55, H-4), 6.94 (1H, dd, J=6.35 & 2.60, H-6), 6.72 (1H,s, H-3), 4.61 (2H, s, 2-CH₂), 3.86 (3H, s, OCH₃).

7-((5-methoxybenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (27)VG027-05

Sodium ethoxide (12 mg, 0.18 mmol) was added to DMF (5 mL) at 0° C., andthe suspension was stirred for 10 min. 7-hydroxy-4-methylcoumarin (32mg, 0.17 mmol) was slowly added and resulting mixture was stirred atthis temp for 0.5 h. To this mixture 26 (40 mg, 0.17 mmol) was addedportionwise. Resulting reaction mixture was stirred at room temperaturefor 2 h. DMF was evaporated off in-vacuo and the residue was purified byflash chromatography, eluting with hexane: EtOAc (3:1) to give 27 (17mg, 30%) as a white solid. m/z=337.04 (M+H), 673.13 (2M+H).

7-((7-methoxybenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (31)VG029-05

2-(2,2-diethoxyethoxy)-3-methoxybenzaldehyde (28)

To a stirred suspension containing 2-hydroxy-3-methoxybenzaldehyde (4.0g, 26.3 mmol) and K₂CO₃ (4.36 g, 31.60 mmol) in DMF (15 mL),bromoacetaldehyde diethyl acetal (4.86 mL, 31.60 mmol) was addeddropwise. The mixture was refluxed for 4 h. After cooling, theprecipitate was filtered off and the solvent was evaporated in vacuo.The crude residue was adsorbed on silica gel and purified by flashchromatography eluting with hexane/EtOAc (4:1) to give 28 (2.60 g, 36%).¹H NMR (500 MHz, CDCl₃): δ: 10.53 (1H, s, CHO), 7.42 (1H, m, ArH),7.14-7.12 (2H, m, ArH), 4.83 (1H, t, J=5.30 Hz, CH), 4.21 (2H, d, J=5.35Hz, CH₂), 3.90 (3H, s, OCH₃), 3.74-3.71 (2H, m, CH₂CH₃), 3.60-3.57 (2H,m, CH₂CH₃), 1.22 (6H, t, J=7.05 Hz, 2×CH₃).

7-methoxybenzofuran-2-carbaldehyde (29)

A stirred solution of 28 (2.0 g, 7.46 mmol) in acetic acid (10 mL) wasrefluxed for 24 h. After cooling, the solution was evaporated todryness. The crude product was adsorbed on silica gel and purified byflash chromatography, eluting with hexane/EtOAc (4:1) to give 29 (450mg, 34%) as a white solid. ¹H NMR (500 MHz, CDCl₃): δ: 9.89 (1H, s,CHO), 7.55 (1H, s, ArH), 7.30 (1H, d, J=6.95 Hz, ArH), 7.24 (1H, t,J=7.85, ArH), 6.97 (1H, d, J=6.90 Hz), 4.02 (3H, s, OCH₃).

2-(bromomethyl)-7-methoxybenzofuran (30)

Compound 29 (450 mg, 2.56 mmol) was dissolved in EtOH (10 mL). NaBH₄(104 mg, 2.81 mmol) was added portionwise at 0° C., with vigorousstirring. The suspension was stirred at 0° C. for 15 min and then atroom temperature for 1.5 h. Solvent was evaporated off in-vacuo. Theresulting crude alcohol residue was dissolved in toluene (5 mL) and thesolution was cooled to 0° C. PBr₃ (240 μL, 2.56 mmol) was added dropwiseover 10 min. The reaction mixture was then brought up to roomtemperature and stirred for 1 h. The solvent was evaporated offin-vacuo. The residue was adsorbed on silica gel and purified by flashchromatography, eluting with hexane/EtOAc (4:1) to give 30 (150 mg, 24%)as an oil. ¹H NMR (500 MHz, CDCl₃): δ: 7.19-7.17 (2H, m, ArH), 6.85 (1H,d, J=5.60, ArH), 6.78 (1H, s, ArH), 4.62 (2H, s, 2-CH₂), 4.04 (3H, s,OCH₃).

7-((7-methoxybenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (31)VG029-05

Sodium ethoxide (12 mg, 0.18 mmol) was added to DMF (5 mL) at 0° C., andthe suspension was stirred for 10 min. 7-hydroxy-4-methylcoumarin (32mg, 0.17 mmol) was slowly added and resulting mixture was stirred atthis temp for 0.5 h. To this mixture 30 (40 mg, 0.17 mmol) was addedportionwise. Resulting reaction mixture was stirred at room temperaturefor 2 h. DMF was evaporated off in-vacuo and the residue was purified byflash chromatography, eluting with hexane: EtOAc (3:1) to give 31 (14mg, 25%) as a white solid m/z 337.04 (M+H), 673.13 (2M+H).

7-((5-bromobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (32)VG035-04

7-((5-bromobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (VG035-04)

Sodium ethoxide (76 mg, 1.10 mmol) was added to DMF (10 mL) at 0° C.,and the suspension was stirred for 10 min. 7-hydroxy-4-methylcoumarin(215 mg, 1.22 mmol) was slowly added and resulting mixture was stirredat this temp for 0.5 h. To this mixture5-bromo-2-(chloromethyl)benzofuran (250 mg, 1.02 mmol) was addedportionwise. Resulting reaction mixture was stirred at room temperaturefor 2 h. DMF was evaporated off in-vacuo and the residue was purified byflash chromatography, eluting with hexane: EtOAc (3:1) to give 32 (120mg, 31%) as a white solid. m/z 386 (M+H). H¹ NMR (500 MHz, DMSO-d₆):=7.91 (1H, d, J=2.0 Hz, ArH), 7.71 (1H, d, J=8.80 Hz, ArH), 7.61 (1H, d,J=8.75, ArH), 7.49 (1H, dd, J=6.70 & 2.05 Hz, ArH), 7.20 (1H, d, J=2.45Hz, ArH), 7.13 (1H, s, ArH), 7.09 (1H, dd, J=6.30 & 2.50, ArH), 6.25(1H, s, CH), 5.43 (2H, s, CH₂), 2.41 (3H, s, CH₃). ¹³C NMR CDEPT 135,(500 MHz, CDCl₃): δ: 127.55 (ArCH), 126.59 (ArCH), 124.04 (ArCH), 113.32(ArCH), 112.56 (ArCH), 111.44 (ArCH), 106.87 (ArCH), 101.66 (ArCH),62.40 (CH₂), 16.11 (CH₃).

7-((5-chlorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (36)VG028-05

5-chloro-2-(2,2-diethoxyethoxy)benzaldehyde (33)

To a stirred suspension of 5-chloro-2-hydroxybenzaldehyde (5.0 g, 32.1mmol) and K₂CO₃ (4.87 g, 35.3 mmol) in DMF (20 mL), bromoacetaldehydediethyl acetal (5.43 mL, 35.3 mmol) was added dropwise. The mixture wasrefluxed for 4 h. After cooling, the precipitate was filtered off andthe solvent was evaporated in vacuo. The crude residue was adsorbed onsilica gel and purified by flash chromatography. The product was elutedwith hexane/EtOAc (4:1) to give 33 (4.10 g, 38%) as an oil. ¹H NMR (500MHz, CDCl₃): δ: 10.42 (1H, s, CHO), 7.76 (1H, d, J=2.8 Hz, ArH), 7.46(1H, dd, J=6.15 & 2.75 Hz, ArH), 6.96 (1H, d, J=8.90 Hz, ArH), 4.87 (1H,t, J=5.25 Hz, CH), 4.10 (2H, d, J=5.25 Hz, CH₂), 3.80-3.77 (2H, m,CH₂CH₃), 3.67-3.62 (2H, m, CH₂CH₃), 1.24 (6H, t, J=7.05 Hz, 2×CH₃).

5-chlorobenzofuran-2-carbaldehyde (34)

A stirred solution of 33 (4.10 g, 15.07 mmol) in acetic acid (20 mL) wasrefluxed for 24 h. After cooling, the solution was evaporated todryness. The crude product was adsorbed on silica gel and purified byflash chromatography, eluting with hexane/EtOAc (4:1) to give 34 (550mg, 20%) as a white solid. ¹H NMR (500 MHz, CDCl₃): δ: 9.91 (1H, s,CHO), 7.76 (1H, d, J=1.85 Hz, ArH), 7.57 (1H, d, J=8.90 Hz, ArH), 7.53(1H, s, ArH), 7.50 (1H, dd, J=8.90 & 2.10 Hz, ArH).

2-(bromomethyl)-5-chlorobenzofuran (35)

Compound 34 (160 mg, 0.89 mmol) was dissolved in EtOH (5 mL). NaBH₄ (36mg, 0.98 mmol) was added portionwise at 0° C., with vigorous stirring.The suspension was stirred at 0° C. for 15 min and then at roomtemperature for 1.5 h. Solvent was evaporated off in-vacuo. Theresulting crude alcohol residue was dissolved in toluene (5 mL) and thesolution was cooled to 0° C. PBr₃ (92 μL, 0.98 mmol) was added dropwiseover 10 min. The mixture was then brought up to room temperature andstirred for 1 h. The solvent was evaporated off in-vacuo. The residuewas adsorbed on silica gel and purified by flash chromatography, elutingwith hexane/EtOAc (4:1) to give 35 (128 mg, 57%) as an oil. ¹H NMR (500MHz, CDCl₃): δ: 7.48 (1H, d, J=2.05 Hz, ArH), 7.37 (1H, d, J=8.70, ArH),7.25 (1H, dd, J=8.80 & 2.05 Hz ArH), 6.68 (1H, s, ArH), 4.55 (2H, s,2-CH₂).

7-((5-chlorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (36)VG028-05

Sodium ethoxide (60 mg, 0.24 mmol) was added to DMF (5 mL) at 0° C., andthe suspension was stirred for 10 min. 7-hydroxy-4-methylcoumarin (47mg, 0.27 mmol) was slowly added and resulting mixture was stirred atthis temp for 0.5 h. To this mixture 2-(bromomethyl)-5-methoxybenzofuran(40 mg, 0.17 mmol) was added portionwise. Resulting reaction mixture wasstirred at room temperature for 2 h. DMF was evaporated off in-vacuo andthe residue was purified by flash chromatography, eluting with hexane:EtOAc (3:1) to give the target compound as a white solid (2.7 mg, 3%).m/z 341.10 (M+H).

7-((5,7-dimethoxybenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (42)VG035-05

2-Bromo-3,5-dimethoxybenzaldehyde (37)

3,5-dimethoxybenzaldehyde (12.6 g, 76 mmol) was dissolved in acetic acid(350 mL). The resulting colourless solution was cooled to 0° C. Asolution of bromine (3.9 mL) in acetic acid (50 mL) was added dropwiseover 1 h. Once the addition was complete the ice bath was removed andthe resulting pale green solution was stirred overnight at roomtemperature. Cold water was added to the solution. The resulting whitesolid was collected by vacuum filteration and rinsed with water. Thesolid was then redissolved in EtOAc and adsorbed on silica gel. Theproduct was purified by flash chromatography, eluting with hexane/EtOAc(4:1) to give 37 (12.5 g, 66%) as a white solid. m/z=344.98 (M+H). ¹HNMR (500 MHz, CDCl₃): δ: 10.43 (1H, s, CHO), 7.06 (1H, s, ArH), 6.73(1H, s, ArH), 3.93 (3H, s, CH₃O), 3.86 (3H, s, CH₃O). ¹³C NMR (500 MHz,CDCl₃): δ: 192.09 (CHO), 159.92 (C-5), 157.02 (C-3), 134.67 (C-1),109.12 (C-2), 105.83, 103.37 (C-4 & C-6), 56.60 (OMe), 55.82 (OMe).

2-Hydroxy-3,5-dimethoxybenzaldehyde (38)

Morpholine (2.05 g, 24 mmol) and THF (40 mL) were placed in a dry,three-necked, round bottomed flask equipped with a stirring bar, septumcap, dropping funnel, thermometer, and argon inlet. The flask was cooledin a dry ice-acetone bath to −50° C., and a solution of n-BuLi in hexane(1.6M, 15 mL, 24 mmol) was added all at once. After 10 min a solution ofthe 2-bromo-3,5-dimethoxybenzadehyde 37 (4.9 g, 20 mmol) in THF (30 mL)was added dropwise via a syringe over a period of 4 min, and the mixturewas cooled to ˜−75° C. over 20 min. n-BuLi in hexane (1.6M, 20 mL, 32mmol) was then added dropwise over 45 min, keeping the temperature at−75° C. After complete addition of n-BuLi the solution was stritted for35 min. A solution of nitrophenol (6.90 g, 46 mmol) in 10 mL THF wasadded from the dropping funnel, keeping the temperature at −75° C. Theresulting dark mixture was stirred at −75° C. for 4 h and then allowedto warm to room temperature. It was acidified to pH 1 with 6N HCl andstirred for 15 min. After dilution with brine (100 mL), THF was removedin-vacuo. The aqueous solution was extracted with diethyl ether (4×40mL). The combined organic layers were extracted with 2 N NaOH (3×40 mL).The combined NaOH extracts were washed with diethyl ether (3×20 mL) andthen acidified to pH 1 with concentrated HCl. The resulting mixture wasextracted with CH₂Cl₂ (3×20 mL), and the combined organic extracts werewashed with brine, dried (MgSO₄) and adsorbed on silica gel. The productwas purified by flash chromatography, eluting with EtOAc/hexane (1:2) togive 38 (2.0 g, 55%) as a yellow solid. m/z=183.06 (M+H). ¹H NMR (500MHz, CDCl₃): δ: 10.71 (1H, s, OH), 9.91 (1H, s, CHO), 6.77 (1H, d,J_(4, 6)=2.8 Hz, H-6), 6.61 (1H, d, J_(4, 6)=2.8 Hz, H-4), 3.92 (3H, s,OMe), 3.84 (3H, s, OMe). ¹³C NMR CDEPT135 (500 MHz, CDCl₃): δ: 196.11(CHO), 107.93 (C-6), 103.90 (C-4), 56.29 (OMe), 55.83 (OMe).

2-(2,2-Diethoxyethoxy)-3,5-dimethoxybenzaldehyde (39)

To a stirred suspension containing 38 (1.1 g, 6.0 mmol) and K₂CO₃ (1.0g, 7.2 mmol) in DMF (100 mL), bromoacetaldehyde diethyl acetal (0.93 mL,6.0 mmol) was added dropwise. The mixture was refluxed for 4 h. Aftercooling, the precipitate was filtered off and the solvent was evaporatedin vacuo. The crude residue was adsorbed on silica gel and purified byflash chromatography. The product was eluted with hexane/EtOAc (4:1) togive 39 (1.2 g, 67%) as an oil. ¹H NMR (500 MHz, CDCl₃): δ: 10.50 (1H,s, CHO), 6.88 (1H, d, J_(4, 6)=2.9 Hz, H-6), 6.74 (1H, d, J_(4, 6)=2.9Hz, H-4), 4.83 (1H, t, J_(4, 6)=5.3 Hz, CH), 4.14 (2H, d, J_(4, 6)=5.3Hz, CH₂), 3.88 (3H, s, OMe), 3.83 (3H, s, OMe), 3.77-3.71) (2H, m,CH₂CH₃), 3.63-3.58 (2H, m, CH₂CH₃), 1.24 (6H, t, J=7.1 Hz, 2×CH₃).

5,7-dimethoxybenzofuran-2-carbaldehyde (40)

A stirred solution of 39 (1.2 g, 4.0 mmol) in acetic acid (35 mL) wasrefluxed for 16 h. After cooling, the solution was evaporated todryness. The crude product was adsorbed on silica gel and purified byflash chromatography, eluting with hexane/EtOAc (2:1) to give 40 (230mg, 28%) as a white solid. ¹H NMR (500 MHz, CDCl₃): δ: 9.89 (1H, s,CHO), 7.50 (1H, s, H-3), 6.69 (1H, d, J_(4, 6)=2.2 Hz, H-6), 6.64 (1H,d, J_(4, 6)=2.2 Hz, H-4), 4.01 (3H, s, OMe), 3.87 (3H, s, OMe). ¹³C NMRCDEPT 135, (500 MHz, CDCl₃): δ: 179.91 (CHO), 153.37 (C-2), 116.10(C-3), 101.80 (C-6), 94.90 (C-4), 56.20 (OMe), 55.88 (OMe).

(5,7-dimethoxybenzofuran-2-yl)methanol (41)

Compound 39 (460 mg, 2.23 mmol) was dissolved in THF (5 mL) and EtOH (1mL). NaBH₄ (102 mg, 2.68 mmol) was added portionwise at 0° C., withvigorous stirring. The suspension was stirred at 0° C. for 15 min andthen at room temperature for 1 h. Solvents were evaporated off in-vacuo.The crude residue was taken up in EtOAc and washed with water, brine anddried (MgSO₄). The residue was adsorbed on silica gel and purified byflash chromatography, eluting with hexane/EtOAc (1:1) to give 41 (388mg, 82%) as a white solid. m/z=209.08 (M+H). ¹H NMR (500 MHz, CDCl₃): δ:6.62 (1H, s, H-3), 6.60 (1H, s, H-6), 6.46 (1H, s, H-4), 4.76 (2H, s,2-CH₂), 3.99 (3H, s, OMe), 3.85 (3H, s, OMe). ¹³C NMR (500 MHz, CDCl₃):δ: 157.23 (C-5), 156.72 (C-1), 145.36 (C-7), 139.50 (C-1a), 129.38(C-4a), 104.62 (C-3), 96.96 (C-6), 94.58 (C-4), 57.98 (2-CH₂), 55.95(OMe), 55.83 (OMe).

7-((5,7-dimethoxybenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (42)VG035-05

Compound 41 (130 mg, 0.63 mmol) was dissolved in toluene (5 mL) and thesolution was cooled to 0° C. PBr₃ (64 μL, 0.69 mmol) was added dropwiseover 10 min. The reaction mixture was then brought up to roomtemperature and stirred for 1 h. The solvent was evaporated offin-vacuo. The crude residue was used in the next step. Sodium ethoxide(80 mg, 0.24 mmol) was added to DMF (5 mL) at 0° C., and the suspensionwas stirred for 10 min. 7-hydroxy-4-methylcoumarin (47 mg, 0.27 mmol)was slowly added and resulting mixture was stirred at this temp for 0.5h. To this mixture 2-(bromomethyl)-5-methoxybenzofuran (40 mg, 0.17mmol) was added portionwise. Resulting reaction mixture was stirred atroom temperature for 2 h. DMF was evaporated off in-vacuo and theresidue was purified by flash chromatography, eluting with hexane: EtOAc(3:1) to give 42 (2.7 mg, 3%) as a white solid. m/z=367.05 (M+H), 733.15(2M+H).

4-methyl-7-(naphthalene-1-ylmethoxy)-2H-chromen-2-one (43) TLE-M1-SU001A

1-Naphthalene methanol (2.0 g, 12.7 mmol) was dissolved in toluene (30mL) and pyridine (1.02 mL, 12.7 mmol) was added. The solution was cooledto 0° C. PBr₃ (1.19 mL, 12.7 mmol) was added dropwise over 15 min. Thereaction mixture was then brought up to room temperature and stirred for1 h. The mixture was washed with K₂CO₃ solution and extracted with EtOAc(3×30 mL). The EtOAc layer was washed with brine and dried (MgSO₄). Thesolvent was evaporated off in-vacuo to give 1-(bromomethyl)naphthalene(1.5 g, 53%) as a colourless oil, This intermediate was used in thefollowing reaction.

Sodium ethoxide (169 mg, 2.49 mmol) was added to DMF (5 mL) at 0° C.,and the suspension was stirred for 10 min. 7-hydroxy-4-methylcoumarin(438 mg, 2.49 mmol) was slowly added and resulting mixture was stirredat this temp for 0.5 h, then allowed to reach room temperature. To thismixture 1-(bromomethyl)naphthalene (500 mg, 2.26 mmol) was addedportionwise. Resulting reaction mixture was stirred at room temperaturefor 16 h. DMF was evaporated off in-vacuo and the residue was taken upin EtOAc, and washed with brine (2×50 mL), water (2×50 mL) and 1M NaOH(2×30 mL). The organic layer was dried (MgSO₄) and product was purifiedby flash chromatography, eluting with hexane:EtOAc (2:1) to give 43 (200mg, 28%) as a white solid. Mpt=181-183° C. H¹ NMR (500 MHz, acetone-d₆):δ=8.10 (1H, d, ArH), 8.00-7.99 (2H, m, Ar), 7.97-7.71 (2H, m, ArH),7.70-7.53 (3H, m, ArH), 7.24 (1H, s, ArH), 7.09 (1H, d, CH), 6.23 (1H,s, CH), 5.68 (2H, s, CH₂), 2.40 (3H, s, CH₃).

4-methyl-7-(naphthalen-2-ylmethoxy)-2H-chromen-2-one (44) VG040-03

Naphthalen-2-ylmethanol (2.0 g, 12.7 mmol) was dissolved in toluene (30mL) and pyridine (1.02 mL, 12.7 mmol) was added. The solution was cooledto 0° C. PBr₃ (1.19 mL, 12.7 mmol) was added dropwise over 15 min. Themixture was then brought up to room temperature and stirred for 1 h. Themixture was washed with K₂CO₃ solution and extracted with EtOAc (3×30mL). The EtOAc layer was washed with brine and dried (MgSO₄). Thesolvent was evaporated off in-vacuo to give crude1-(bromomethyl)naphthalene. This intermediate was used in the followingreactions.

Sodium ethoxide (169 mg, 2.49 mmol) was added to DMF (5 mL) at 0° C.,and the suspension was stirred for 10 min. 7-hydroxy-4-methylcoumarin(438 mg, 2.49 mmol) was slowly added and resulting mixture was stirredat this temp for 0.5 h, then allowed to reach room temp. To this mixture1-(bromomethyl)naphthalene (500 mg, 2.26 mmol) was added portionwise.Resulting reaction mixture was stirred at room temperature for 16 h. DMFwas evaporated off in-vacuo and the residue was taken up in EtOAc, andwashed with brine (2×50 mL), water (2×50 mL) and 1M NaOH (2×30 mL). Theorganic layer was dried (MgSO₄) and product was purified by flashchromatography, eluting with hexane: EtOAc (2:1) to give 44 (1.44 g,36%) as a white solid. m/z=317.12 (M+H), 633.24 (2M+H). H¹ NMR (500 MHz,CDCl₃): δ=7.93-7.87 (4H, m, ArH), 7.58-7.52 (4H, m, Ar), 6.97 (1H, t,J=2.43 Hz, ArH), 6.16 (1H, s, ArH), 5.32 (2H, s, CH₂), 2.41 (3H, s,CH₃).

7-(benzhydryloxy)-4-methyl-2H-chromen-2-one (45) TLE-M1-SU004A

Sodium ethoxide (165 mg, 2.43 mmol) was added to DMF at 0° C., and thesuspension was stirred for 10 min. 7-hydroxy-4-methylcoumarin (428 mg,2.43 mmol) was slowly added and resulting mixture was stirred at thistemp for 0.5 h, then allowed to reach room temp. To this mixturediphenylmethyl bromide (500 mg, 2.02 mmol) was added portionwise.Resulting reaction mixture was stirred at room temperature for 16 h. DMFwas evaporated off in-vacuo and the residue was taken up in EtOAc, andwashed with brine (2×30 mL), water (2×30 mL) and 1M NaOH (2×30 mL). Theorganic layer was dried (MgSO₄) and product was purified by flashchromatography, eluting with hexane: EtOAc (2:1) to give 45 (200 mg,29%) as a white solid. Mpt=146-148° C. H¹ NMR (500 MHz, DMSO-d₆): δ=7.63(1H, d, ArH), 7.53 (4H, d, ArH), 7.38 (4H, t, ArH), 7.29 (2H, t, ArH),7.09 (2H, d, ArH), 7.04 (1H, d, ArH), 6.75 (1H, s, ArH), 6.18 (1H, s,CH), 2.37 (3H, s, CH₃). ¹³C NMR (500 MHz, DMSO-d₆, DEPT135): δ=160.2,154.4, 153.2, 140.8, 129.90, 128.0, 126.8, 113.7, 111.4, 111.2, 102.9,80.2, 18.2.

4-methyl-7-(1-(naphthalen-2-yl)ethoxy)-2H-chromen-2-one (46) VG039-03

1-(Naphthalen-2-yl)ethanol (2.0 g, 11.6 mmol) was dissolved in toluene(30 mL). The solution was cooled to 0° C. PBr₃ (1.09 mL, 11.6 mmol) wasadded dropwise over 15 min. The reaction mixture was then brought up toroom temperature and stirred for 1 h. The mixture was washed with K₂CO₃solution and extracted with EtOAc (3×30 mL). The EtOAc layer was washedwith brine and dried (MgSO₄). The solvent was evaporated off in-vacuo togive crude 2-(1-bromoethyl)naphthalene. This intermediate was used inthe following step.

Sodium ethoxide (63.4 mg, 0.93 mmol) was added to DMF (5 mL) at 0° C.,and the suspension was stirred for 10 min. 7-hydroxy-4-methylcoumarin(147 mg, 0.84 mmol) was slowly added and resulting mixture was stirredat this temperature for 0.5 h, then allowed to reach room temperature.To this mixture 2-(1-bromoethyl)naphthalene (200 mg, 0.85 mmol) wasadded portionwise. Resulting reaction mixture was stirred at roomtemperature for 16 h. DMF was evaporated off in-vacuo and the residuewas taken up in EtOAc, and washed with brine (2×50 mL), water (2×50 mL)and 1M NaOH (2×30 mL). The organic layer was dried (MgSO₄) and productwas purified by flash chromatography, eluting with hexane: EtOAc (2:1)to give 46 (60 mg, 21%) as a white solid. m/z=331.15 (M+H), 661.29(2M+H). H¹ NMR (500 MHz, DMSO-d₆): δ=7.97 (1H, s, ArH), 7.93-7.86 (3H,m, ArH), 7.60-7.50 (4H, m, Ar), 6.99 (1H, q, J=6.45 & 2.35 Hz, ArH),6.96 (1H, d, J=2.40 Hz, ArH), 6.13 (1H, s, ArH), 5.84 (1H, q, J=6.35 Hz,CH), 2.26 (3H, s, CH₃), 1.67 (3H, d, J=6.35 Hz, CH₃).

7-(anthracen-9-ylmethoxy)-4-methyl-2H-chromen-2-one (48) SU06-02

9-(bromomethyl)anthracene (47)

To a stirring suspension of 9-anthracenemethanol (2.0 g, 9.6 mmol) at 0°C. in toluene (100 ml) was added PBr₃ (1.2 mL, 12.51 mmol) and thesuspension was stirred at 0° C. for 1 h. The reaction mixture was thenbrought up to room temperature and let to stir for further 1 h. Themixture turned into a yellow solution. K₂CO₃ (10 mL) was added to quenchthe reaction. Toluene was evaporated off in-vacuo. The residue was takenup in EtOAc and washed with saturated aqueous K₂CO₃, water and brine anddried (MgSO₄). The solvent was evaporated off in-vacuo and the cruderesidue was purified by flash chromatography, eluting with hexane:EtOAc(2:1) to give 47 (1.4 g, 54%) as yellow solid. H¹ NMR (500 MHz, CDCl₃):δ=8.45 (1H, s, Ar-10H), 8.27 (2H, d, Ar-1,8 H), 8.00 (2H, d, Ar-4, 6H),7.62 (2H, d, Ar-2, 7H), 7.48 (2H, d, Ar-3, H), 5.50 (2H, s, CH₂).

7-(anthracen-9-ylmethoxy)-4-methyl-2H-chromen-2-one (48) SU06-02

Sodium ethoxide (151 mg, 2.21 mmol) was added to DMF (5 mL) at 0° C.,and the suspension was stirred for 10 min. 7-hydroxy-4-methylcoumarin(390 mg, 2.21 mmol) was slowly added and resulting mixture was stirredat this temp for 0.5 h, then allowed to reach room temp. To this mixture47 (500 mg, 1.85 mmol) was added portionwise. Resulting reaction mixturewas stirred at room temperature for 16 h. DMF was evaporated offin-vacuo and the residue was taken up in EtOAc, and washed with brine,water and 1M NaOH (2×30 mL). The organic layer was dried (MgSO₄) andproduct was purified by flash chromatography, eluting with hexane: EtOAc(2:1) to give 48 (200 mg, 30%) as a yellow solid. Mpt=216-218° C. H¹ NMR(500 MHz, DMSO-d₆): δ=8.10 (1H, d, ArH), 8.00-7.99 (2H, m, Ar),7.97-7.71 (2H, m, ArH), 7.70-7.53 (3H, m, ArH), 7.24 (1H, s, ArH), 7.09(1H, d, CH), 6.23 (1H, s, CH), 5.68 (2H, s, CH₂), 2.40 (3H, s, CH₃). ¹³CNMR (500 MHz, DMSO-d₆, DEPT135): δ=160.8 (qC), 152.0 (2×qC), 129.0(2×CH), 128.9 (2×CH), 126.8, (2×CH), 126.8 (Ar CH), 126.5 (Ar CH), 125.3(Ar CH), 124.1 (Ar CH), 112.9 (coumarin 3-CH), 111.2 (coumarin 6-CH),101.7 (coumarin 8-CH), 62.9 (CH₂), 18.2 (CH₃).

7-(bis(4-methoxyphenyl)methoxy)-4-methyl-2H-chromen-2-one (49) SU010-02

Bis(4-methoxyphenyl)methanol (2.0 g, 8.2 mmol) was dissolved in toluene(60 mL) and pyridine (661 μL, 8.2 mmol)) was added. The solution wascooled to 0° C. PBr₃ (768 μL, 8.2 mmol) was added dropwise over 15 min.The reaction mixture was warmed to room temperature and stirred for 1 h.The mixture was washed with K₂CO₃ solution and extracted with EtOAc(3×30 mL). The EtOAc layer was washed with brine and dried (MgSO₄). Thesolvent was evaporated off in-vacuo to give the crude product4,4′-(bromomethylene)bis(methoxybenzene) (780 mg, 31%), as a colourlessoil. This was used in the next reaction step without furtherpurification. Sodium ethoxide (133 mg, 1.96 mmol) was added to DMF (5mL) at 0° C., and the suspension was stirred for 10 min.7-hydroxy-4-methylcoumarin (345 mg, 1.96 mmol) was slowly added andresulting mixture was stirred at this temp for 0.5 h. To this mixture4,4′-(bromomethylene)bis(methoxybenzene (500 mg, 1.63 mmol) was addedportionwise. Resulting reaction mixture was stirred at room temperaturefor 16 h. DMF was evaporated off in-vacuo and the residue was taken upin EtOAc, and washed with brine, water and 1M NaOH (2×30 mL). Theorganic layer was dried (MgSO₄) and product was purified by flashchromatography, eluting with hexane: EtOAc (2:1) to give 49 (100 mg,15%) as a white solid. Mpt=142-145° C. m/z=403 (M+H). H¹ NMR (500 MHz,acetone-d₆): δ=7.60 (1H, d, ArH), 7.45 (4H, d, ArH), 7.04 (1H, d, ArH),6.94 (5H, d, ArH), 6.56 (1H, s ArH), 6.10 (1H, s, qCH), 3.78 (6H, s,2×CH₃O), 2.39 (3H, s, CH₃). ¹³C NMR (500 MHz, acetone-d₆, DEPT135):δ=206.3 (qC), 134.1 (2×qC), 129.3 (2×CH), 129.2 (2×CH), 129.0, (2×CH),126.9 (Ar CH), 114.4 (Ar CH), 114.7 (Ar CH), 115.1 (Ar CH), 112.5 (ArCH), 104.0 (Ar CH), 81.7 (CH), 55.6 (2×CH₃), 18.2 (CH₃).

7-((1H-benzo[d]imidazol-2-yl)methoxy)-4-methyl-2H-chromen-2-one (50)VG033-03

Sodium ethoxide (82 mg, 1.20 mmol) was added to DMF (10 mL) at 0° C.,and the suspension was stirred for 10 min. 7-hydroxy-4-methylcoumarin(253 mg, 1.44 mmol) was slowly added and resulting mixture was stirredat this temp for 0.5 h. To this mixture2-(chloromethyl)-1H-benzo[d]imidazole (200 mg, 1.20 mmol) was addedportionwise. Resulting reaction mixture was stirred at room temp for 2h. DMF was evaporated off in-vacuo and the residue was purified by flashchromatography, eluting with hexane:EtOAc (3:1) to give 50 (200 mg, 54%)as a white solid. m/z=307.11 (M+H), 613.22 (2M+H). H¹ NMR (500 MHz,DMSO-d₆): δ=12.75 (1H, brs, NH), 7.74 (1H, d, J=8.85 Hz, ArH), 7.60-7.59(2H, m, ArH), 7.23-7.12 (4H, m, ArH), 6.25 (1H, s, ArH), 5.48 (2H, s,CH₂), 2.40 (3H, s, CH₃). ¹³C NMR CDEPT 135, (500 MHz, CDCl₃): δ: 206.52(qC), 160.80, 160.03, 154.52, 153.33, 149.27, 126.57, 126.28, 122.04,119.42, 113.64, 112.50, 111.47, 101.86, 101.77, 64.23, 30.67, 18.10.

7-(benzo[d]thiazol-2-ylmethoxy)-4-methyl-2H-chromen-2-one (51) VG014-04

Sodium ethoxide (30 mg, 0.44 mmol) was added to DMF (10 mL) at 0° C.,and the suspension was stirred for 10 min. 7-hydroxy-4-methylcoumarin(77 mg, 0.44 mmol) was slowly added and resulting mixture was stirred atthis temp for 0.5 h. To this mixture2-(chloromethyl)-1H-benzo[d]imidazole (100 mg, 0.44 mmol) was addedportionwise. Resulting reaction mixture was stirred at room temperaturefor 2 h. DMF was evaporated off in-vacuo and the residue was purified byflash chromatography, eluting with hexane: EtOAc (3:1) to give 51 (25mg, 18%) as a white solid. m/z=324.06 (M+H), 647.12 (2M+H).

4-methyl-7-(4-(thiophen-2-yl)benzyloxy)-2H-chromen-2-one (52) VG015-04

Sodium ethoxide (27 mg, 0.40 mmol) was added to DMF (10 mL) at 0° C.,and the suspension was stirred for 10 min. 7-hydroxy-4-methylcoumarin(70 mg, 0.40 mmol) was slowly added and resulting mixture was stirred atthis temp for 0.5 h. To this mixture2-(chloromethyl)-1H-benzo[d]imidazole (100 mg, 0.40 mmol) was addedportionwise. Resulting reaction mixture was stirred at room temp for 2h. DMF was evaporated off in-vacuo and the residue was purified by flashchromatography, eluting with hexane: EtOAc (3:1) to give 52 (30 mg, 18%)as a white solid. m/z=349.09 (M+H), 697.16 (2M+H).

6-(benzhydrylthio)-9H-purine (53) (VG015-02)

6-Mercaptopurine (151 mg, 0.88 mmol) was dissolved in DMF (5 mL). K₂CO₃(122 mg, 1.2 mmol) was added and to the resulting suspension, diphenylmethylbromide (200 mg, 0.8 mmol) was added. The resulting reactionmixture was stirred at room temperature for 4 h. The mixture was pouredon ice and the resulting precipitate was separated by filteration,washed with ether and dried in vacuo to give 53 (35 mg, 14%) as a whitesolid. m/z=319 (M+H). H¹ NMR (500 MHz, acetone): δ=8.46 (1H, s, CH), 8.2(1H, s, CH), 7.4 (4H, m, CH, J=3), 7.2 (4H, m, CH, J=3.83), 7.1 (2H, m,CH, J=2.12), 6.7 (1H, s, CH).

3. Carbamate-Linked Nucleoside Analogue Prodrugs

SU001- 03

SU0044- 2a/02 (†)

SU0023/ 02

SU0044- 3a/02 (†)

SU050- 03

SU048- 04

naphthalen-1-ylmethyl1-(3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-ylcarbamate(55) SU001-03

Naphthalen-1-ylmethanol (3.0 g, 19.0 mmol) was added in one portion toCOCl₂ (13.3 mL, as 20% solution of COCl₂ in toluene) in THF (30 mL). Thereaction was stirred at room temperature for 2 h. Excess COCl₂ and THEwas removed under reduced pressure. The solid residue was dissolved inhot hexane and filtered. The hexane was then slowly evaporated off invacuo to obtain the chloroformate intermediate 54 as a white solid. Thiswas used straight away in the following step. 54 (330 mg, 1.5 mmol) andKHCO₃ (252 mg, 2.52 mmol) were added to a solution of cytarabine.HCl(243 mg, 0.87 mmol) in dimethyl acetamide (5 mL), and the mixture wasstirred for 16 h at room temperature. The solvent was evaporated off invacuo and the product was purified by flash chromatography, eluting witha gradient of 2.5%-12% MeOH in DCM to obtain 55 (38 mg, 10%) as a whitesolid. m/z=428.15 (M+H).

naphthalen-1-ylmethyl1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-ylcarbamate(56) SU0023-02

Naphthalen-1-ylmethanol (1.0 g, 6.3 mmol) was added in one portion toCOCl₂ (4.4 mL, as 20% solution of COCl₂ in toluene) in THF (20 mL). Thereaction was stirred at room temperature for 2 h. Excess COCl₂ and THFwas removed under reduced pressure. The solid residue was dissolved inhot hexane and filtered. The hexane solvent was then slowly evaporatedoff in vacuo to obtain the chloroformate intermediate 54 as a whitesolid. This was used straight away in the following step.Gemcitabine.HCl (200 mg, 0.67 mmol) was dissolved in H₂O (2 mL). To thiswas added KHCO₃ (67 mg, 0.67 mmol) and 54 (147 mg, 0.67 mmol),predissolved in ethyl acetate (5 mL). The mixture was stirred at 100° C.for 16 h. The solvent was evaporated off in vacuo and the product waspurified by flash chromatography, eluting with 3% MeOH in ethyl acetateto obtain 56 (15 mg, 5%) as an oil. m/z=448.13 (M+H).

benzyl1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-ylcarbamate(57) SU0044-2a/02

Gemcitabine.HCl (200 mg, 0.67 mmol) was dissolved in H₂O (2 mL). To thiswas added KHCO₃ (67 mg, 0.67 mmol) and benzyl carbonochloridate (95 μL,0.67 mmol), predissolved in ethyl acetate (5 mL). The mixture wasstirred at 80° C. for 16 h. The solvent was evaporated off in vacuo andthe product was purified by flash chromatography, eluting with 3% MeOHin ethyl acetate to give 57 (40 mg, 15%) as an oil. m/z=398.12 (M+H).

benzyl1-(3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-ylcarbamate(58) SU0044-3a/02

Cytarabine.HCl (200 mg, 0.72 mmol) was dissolved in H₂O (2 mL). To thiswas added KHCO₃ (72 mg, 0.72 mmol) and benzyl carbonochloridate (107 μL,0.72 mmol), predissolved in ethyl acetate (5 mL). The mixture wasstirred at 80° C. for 16 h. The solvent was evaporated off in vacuo andthe product was purified by flash chromatography, eluting with 3% MeOHin ethyl acetate to give 58 (40 mg, 15%) as an oil. m/z=378.13 (M+H).

benzofuran-2-ylmethyl 4-nitrophenyl carbonate (60) and(5,7-dimethoxybenzofuran-2-yl)methyl 4-nitrophenyl carbonate (61)

Benzofuran-2-ylmethyl 4-nitrophenyl carbonate (60)

A solution of benzofuran-2-ylmethanol 59 (300 mg, 2.03 mmol) in THF (5mL) was cooled to 0° C. TEA (280 μL, 2.03 mmol) was added dropwisefollowed by the portionwise addition of p-nitrophenyl chloroformate (282mg, 3.05 mmol). The resulting solution was stirred at room temperaturefor 2 h. Solvent was evaporated off in vacuo and the crude residue waspurified by flash chromatography, eluting with hexane: EtOAc (3:1) togive 60 (350 mg, 54%) as a white solid. H¹ NMR (500 MHz, CDCl₃): δ=8.31(2H, m, ArH), 7.62 (1H, d, J=7.70 Hz, ArH), 7.54 (1H, d, J=7.70 Hz,ArH), 7.43-7.27 (3H, m, ArH), 7.29 (1H, t, J=7.72 Hz, ArH), 6.93 (1H, s,ArH), 5.43 (2H, s, CH₂). ¹³C NMR CDEPT 135, (500 MHz, CDCl₃): δ=125.47,125.37, 123.23, 121.80, 121.66, 111.60, 108.53, 62.95.

(5,7-dimethoxybenzofuran-2-yl)methyl 4-nitrophenyl carbonate (61)

A solution of (5,7-dimethoxybenzofuran-2-yl)methanol 6 (100 mg, 0.48mmol) in THF (3 mL) was cooled to 0° C. TEA (69 uL, 0.48 mmol) was addeddropwise followed by the portionwise addition of p-nitrophenylchloroformate (100 mg, 0.72 mmol). The resulting solution was stirred atroom temperature for 2 h. Solvent was evaporated off in vacuo and thecrude residue was purified by flash chromatography, eluting with hexane:EtOAc (3:1), to give 61 (120 mg, 67%) as a white solid. H¹ NMR (500 MHz,CDCl₃): δ=8.29 (2H, d, J=9.0 Hz, ArH), 7.40 (2H, d, J=9.0 Hz, ArH), 6.82(1H, s, ArH), 6.63 (1H, s, ArH), 6.52 (1H, s, ArH), 5.39 (2H, s, CH₂),4.00 (3H, s, OCH₃), 3.85 (3H, s, OCH₃).

benzofuran-2-ylmethyl1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-ylcarbamate(65) SU050-03 and (5,7-dimethoxybenzofuran-2-yl)methyl1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-ylcarbamate(66) SU048-04

4-amino-1-(9,9-difluoro-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)pyrimidin-2(1H)-one(62)

Gemcitabine.HCl (1.0 g, 3.3 mmol), was stirred in pyridine (10 mL) for10 min (2×5 mL). The pyridine was evaporated off. The pyridine (10 mL)was added and 1,1,3,3,-tetraisopropyldisiloxane (1.17 mL, 3.63 mmol) wasadded dropwise. Resulting mixture was stirred at 100° C. for 16 h. Afurther portion of 1,1,3,3,-tetraisopropyldisiloxane (1 mL) was addedand the mixture was stirred at 120° C. for 1 h. Reaction mixture wascooled to room temperature and solvent was evaporated off in vacuo. Theresulting crude solid was recrystalised from EtOAc/ether (1:1) to give62 (600 mg, 36%) as a white solid. m/z=506.23 (M+H).

benzofuran-2-ylmethyl1-(9,9-difluoro-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)-2-oxo-1,2-dihydropyrimidin-4-ylcarbamate(63)

To a stirred solution of 62 (300 mg, 0.59 mmol) in THF (5 mL) was addedbenzofuran-2-ylmethyl 4-nitrophenyl carbonate (223 mg, 0.71 mmol). Theresulting solution was stirred at 100° C. for 4 days. Solvent wasevaporated off in vacuo and the product was purified by preparative HPLCto give 63 (350 mg, 87%) as an oil. m/z=680.0 (M+H), 1359.49 (2M+H).

benzofuran-2-ylmethyl1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-ylcarbamate(65) SU050-03

Compound 63 (200 mg, 0.29 mmol) was dissolved in THF (1.5 mL). To thiswas added tetra-n-butylammonium fluoride and the resulting solution wasstirred at room temperature for 15 min. Solvent was evaporated off invacuo. The product was purified by flash chromatography, eluting with 5%MeOH in EtOAc to give 65 (30 mg, 23%) as an oil. m/z=438.14 (M+H),874.24 (2M+H).

(5,7-dimethoxybenzofuran-2-yl)methyl1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-ylcarbamate(66) SU048-04

To a stirred solution of 62 (108 mg, 0.21 mmol) in THF (5 mL) was added61 (100 mg, 0.27 mmol). The resulting solution was stirred at 100° C.for 4 days. Solvent was evaporated off in vacuo to give 64 as an oil.This was used in the next step without further purification. Compound 64(100 mg, 0.14 mmol) was dissolved in THF (1.5 mL). To this was addedtetra-n-butylammonium fluoride and the resulting solution was stirred atroom temperature for 15 min. Solvent was evaporated off in-vacuo. Theproduct was purified by flash chromatography, eluting with 5% MeOH inEtOAc to give 66 (18 mg, 26%) as an oil. m/z=498.14 (M+H), 995.29(2M+H). H¹ NMR (500 MHz, acetone-d₆): δ=9.60 (1H, bs, NH), 8.34 (1H, d,J=7.62 Hz, ArH), 7.26 (1H, d, J=9.00 Hz, ArH), 6.92 (1H, s, ArH), 6.71(1H. d, J=2.20 Hz, ArH), 6.56 (1H, d, J=2.20 Hz, ArH), 6.26 (1H, t,J=7.56 Hz, CH), 5.64 (2H, s, CH₂), 4.55-4.45 (1H, m, CH), 4.05-4.02 (2H,m, CH₂), 3.97 (3H, s, OCH₃), 3.91-3.3.87 (1H, m, CH), 3.82 (3H, s,OCH₃), 2.92 (2H, bs, OH).

4. Carbamate-Linked Nitrogen and Aniline Mustard Prodrugs

VG042-04

VG0445-04

benzofuran-2-ylmethyl 4-(bis(2-chloroethyl)amino)phenylcarbamate(VG042-04)

2,2′-(4-nitrophenylazanediyl)diethanol (67)

Diethanolamine (2.70 mL, 2.5 mmol) was added to 1-fluoro-4-nitrobenzene(1.0 g, 7.09 mmol) in DMF (30 mL). The resulting mixture was stirred at140° C. for 3.5 h. The solution was cooled to room temperature andsolvent was evaporated off in vacuo. The residue was dissolved in EtOAc(30 mL) and washed with water (3×10 mL) and brine (3×20 mL) and dried(MgSO₄). Solvent was evaporated off in vacuo and the product waspurified by flash chromatography, eluting with EtOAc to give 67 (400 mg,25%) as a yellow solid. H¹ NMR (500 MHz, CDCl₃): =8.06 (2H, d, J=9.50Hz, ArH), 6.87 (2H, d, J=9.50 Hz, ArH), 4.27 (2H, t, J=5.35 Hz, 2×OH),3.83 (4H, q, J=5.55 & 5.45 Hz, 2×CH₂), 3.74 (4H, t, J=5.62 Hz, 2×CH₂).

N,N-bis(2-(tert-butyldimethylsilyloxy)ethyl)-4-nitroaniline (68)

To a cooled solution of 67 (400 mg, 1.77 mmol) and imidazole (481 mg,7.08 mmol) in DMF (10 mL) was added dropwise tert-butyl dimethyl silylchloride (2.72 mg, 3.54 mmol). The mixture was allowed to reach roomtemperature and stirred for 48 h. The solvent was evaporated off invacuo and the product was purified by flash chromatography, eluting with10% EtOAc in ether to give 68 (200 mg, 25%) as a yellow solid. H¹ NMR(500 MHz, CDCl₃): δ=8.06 (2H, d, J=9.45 Hz, ArH), 6.65 (2H, d, J=9.45Hz, ArH), 3.80 (4H, t, J=5.80 Hz, 2×CH₂), 3.62 (4H, t, J=5.80 Hz,2×CH₂), 0.85 (18H, s, 6×CH₃), −0.01 (12H, s, 4×CH₃).

benzofuran-2-ylmethyl 4-(bis(2-chloroethyl)amino)phenylcarbamate (69)VG042-04

Compound 68 (200 mg, 0.44 mmol) was treated with hydrogen in thepresence of 10% Pd on carbon (20 mg). After 16 h stirring, the mixturewas filtered through Celite and the solvent was evaporated off in vacuoto give the intermediate amino aniline product. This was then reactedwith triphosgene (195 mg, 0.70 mmol) in the presence of triethylamine(260 uL, 0.70 mmol) in THF (15 mL). After 1 h stirring at roomtemperature a white precipitate was filtered off and the solvent wasevaporated off in vacuo to give a crude residue of isocyanate aniline.This was used straight away in the following step.

Isocyanate intermediate was dissolved in THF (10 mL). The solution wascooled to 0° C. Benzofuran-2-ylmethanol 59 (100 mg, 1.35 mmol) was addedand the resulting mixture was stirred at room temperature for 16 h. Themixture was cooled on ice and TBAF (996 μL, 3.38 mmol) was addeddropwise over 5 min. The resulting mixture was allowed to warm to roomtemperature and then stirred for 20 min. THF was evaporated off invacuo. The intermediate was dissolved in pyridine (5 mL) and to this wasadded methane sulphonyl chloride (12.5 μL, 0.16 mmol). The mixture wasstirred at room temperature for 1 h. Pyridine was evaporated off invacuo and the crude product was purified by flash chromatography,eluting with hexane:EtOAc (3:1) to give 69 (5 mg, 2%) as a white solid.m/z=408.07 (M+H), 837.16 (2M+H).

benzofuran-2-ylmethyl bis(2-chloroethyl)carbamate (70) VG045-04

A solution of 60 (200 mg, 0.64 mmol) in pyridine (3 mL) was added to asolution of bis(2-chloroethylamine).hydrochloride (227 mg, 1.28 mmol) inpyridine (25 mL). The mixture was stirred at room temperature for 16 h.DCM (10 mL) was added and the mixture was washed with 2% citric acidsolution (2×50 mL), water (50 mL), brine (50 mL) and dried (MgSO₄).Solvent was evaporated off in vacuo and the product was purified byflash chromatography, eluting with CH₂Cl₂:hexane (2:1) to give 70 (125mg, 62%) as an oil. m/z=338.05 (M+Na). H¹ NMR (500 MHz, CDCl₃): δ=7.60(1H, d, J=7.70 Hz, ArH), 7.51 (1H, d, J=8.05 Hz, ArH), 7.33 (1H, t,J=6.80 Hz, ArH), 7.26 (1H, t, J=6.80 Hz, ArH), 6.79 (1H, s, ArH), 5.28(2H, s, CH₂), 3.72-3.63 (8H, m, 4×CH₂).

5. Ether-Linked Topoisomerase I Inhibitor Prodrug

5,7-Dimethoxybenzofuran-2-yl)methyl-camptothecin (71) SU037-04

Compound 41 (100 mg, 0.48 mmol) was dissolved in toluene (5 mL) and thesolution was cooled to 0° C. PBr₃ (46 μL, 0.48 mmol) was added dropwiseover 10 min. The reaction mixture was then brought up to roomtemperature and stirred for 1 h. The solvent was evaporated offin-vacuo. The crude residue was used in the next step.

Sodium ethoxide (15 mg, 0.22 mmol) was added to DMF (5 mL) at 0° C., andthe suspension was stirred for 10 min. Camptothecin (81 mg, 0.22 mmol)was slowly added and resulting mixture was stirred at this temp for 0.5h. To this mixture, crude residue from previous step,2-(bromomethyl)-5,7-dimethoxybenzofuran (50 mg, 0.18 mmol) was addedportionwise. Resulting mixture was stirred at room temperature for 2 h.DMF was evaporated off in-vacuo and the residue was purified by flashchromatography, eluting with DCM:EtOAc (2:1) to give the target compoundas a white solid (10 mg, 10%). m/z=555.19 (M+H).

6. Ether-Linked Tyrosine Kinase Inhibitor Prodrugs

VG048- 04

SU01- A- 04

SU01-B- 04

SU01- C-04

N-(4-(benzofuran-2-ylmethoxy)quinazolin-2-yl)-4,6,7-trimethylquinazolin-2-amine(72) VG048-04

Sodium ethoxide (3 mg, 0.05 mmol) was added to DMF (2 mL) at 0° C., andthe suspension was stirred for 5 min.2-(4,6-dimethylquinazolin-2-ylamino)quinazolin-4-ol (15 mg, 0.05 mmol)was slowly added and resulting mixture was stirred at this temperaturefor 0.5 h. To this mixture 2-(bromomethyl)benzofuran (16 mg, 0.08 mmol)was added. Resulting mixture was stirred at room temperature for 1 h.DMF was evaporated off in-vacuo to give a crude white solid. This waspurified by washing with cold ether and EtOAc to give 72 (3 mg, 11%) asa white solid. m/z=462.2 (M+H).

7-(benzofuran-2-ylmethoxy)-5-isopropyl-2-methyl-[1,2,4]triazolo[1,5-a]pyrimidine(73) SU01-A-04

Sodium ethoxide (7.2 mg, 0.10 mmol) was added to DMF (2 mL) at 0° C.,and the suspension was stirred for 5 min.5-isopropyl-2-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (20 mg, 0.10mmol) was slowly added and resulting mixture was stirred at thistemperature for 0.5 h. To this mixture 2-(bromomethyl)benzofuran (16 mg,0.08 mmol) was added. Resulting mixture was stirred at room temperaturefor 1 h. DMF was evaporated off in-vacuo to give a crude white solid.This was purified by semi-preparative HPLC to give 73 (6.3 mg, 19%) as awhite solid. m/z=323.13 (M+H), 645.27 (2M+H).

7-(benzofuran-2-ylmethoxy)-2-methyl-5-((4-methylpyrimidin-2-ylthio)methyl)-[1,2,4]triazolo[1,5-a]pyrimidine(74) SU01-B-04

Sodium ethoxide (4.7 mg, 0.07 mmol) was added to DMF (2 mL) at 0° C.,and the suspension was stirred for 5 min.2-methyl-5-((4-methylpyrimidin-2-ylthio)methyl)-[1,2,4]triazolo[15-a]pyrimidin-7-ol(20 mg, 0.07 mmol) was slowly added and resulting mixture was stirred atthis temp for 0.5 h. To this mixture 10 (16 mg, 0.08 mmol) was added.Resulting mixture was stirred at room temperature for 1 h. DMF wasevaporated off in-vacuo to give a crude white solid. This was purifiedby semi-preprative HPLC to give 74 (5.2 mg, 18%) as a white solid.m/z=419.09 (M+H), 837.23 (2M+H).

7-(benzofuran-2-ylmethoxy)-1-(2-fluorobenzyl)-4-methyl-1H-[1,2,3]triazolo[4,5-d]pyridazine(75) SU01-C-04

Sodium ethoxide (5.2 mg, 0.08 mmol) was added to DMF (2 mL) at 0° C.,and the suspension was stirred for 5 min1-(2-fluorobenzyl)-4-methyl-1H-[1,2,3]triazolo[4,5-d]pyridazin-7-ol (20mg, 0.08 mmol) was slowly added and resulting mixture was stirred atthis temp for 0.5 h. To this mixture 2-(bromomethyl)benzofuran (16 mg,0.08 mmol) was added. Resulting mixture was stirred at room temperaturefor 1 h. DMF was evaporated off in-vacuo to give a crude white solid.This was purified by semi-preprative HPLC to give 75 (3 mg, 10%)as awhite solid. m/z=390.07 (M+H), 801.12 (2M+Na).

7. Carbamate-Linked Model Coumarin Prodrugs

TLE- M1- SU001C (†)

SU030- 7-03

SU002102 (†)

SU030- 8-03 (†)

VG032- 03

SU033- 03

VG037- 03

SU018- 03

SU024- 2-03

VG032- 05

SU024- 3-03 (†)

VG036- 05

SU030- 4-03

VG041- 05

7-Isocyanato-4-methylcoumarin (76)

A 200-mL three-neck flask fitted with a dry ice condenser and magneticstirrer was charged with a solution of 20% phosgene in toluene solution(2.0 mL) and dioxane (80 mL). To this mixture was added7-amino-4-methyl-2H-chromen-2-one (2.00 g, 11.4 mmol). The mixture wasstirred at 100° C. for 12 h. The initial yellow colour disappeared and awhite solid precipitated. An additional 20% phosgene in toluene solution(7.0 mL) was added and the mixture heated for an additional 5 h, atwhich time the solution cleared. Excess phosgene and traces of HCl wasremoved by bubbling nitrogen gas through the solution. The cloudysolution was filtered to remove unreacted7-amino-4-methyl-2H-chromen-2-one and concentrated to give 76 (0.5 g,25%) as a white solid. H¹ NMR (500 MHz, CDCl₃): δ=7.50 (1H, d, J=7.40Hz, ArH), 7.46 (2H, s, ArH), 6.20 (1H, s, ArH), 2.35 (3H, s, CH₃): ir(CH₂CL₂) 2314 (N═C═O), 1726 and 1615 cm⁻¹.

naphthalen-1-ylmethyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (77)VG020-02

Naphthalen-1-ylmethanol (56 mg, 0.28 mmol) and 76 (200 mg, 1.27 mmol)were dissolved in THF (2 mL). The resulting mixture was stirred at roomtemperature for 15 min and then at 80° C. for 1 h. THF was evaporatedoff in vacuo. The residue was adsorbed on silica and purified by flashchromatography, eluting with CH₂Cl₂/hexane/EtOAc (1:1:1) to give 77 (3mg, 5%) as a white solid. m/z=360.14 (M+H), 719.27 (2M+H).

(2-chloroquinolin-3-yl)methyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate(78) SU030-7-03

(2-chloroquinolin-3-yl)methanol (100 mg, 0.52 mmol) and 76 (155 mg, 0.77mmol) were dissolved in THF (2 mL). The resulting mixture was stirred atroom temperature for 15 min and then at 80° C. for 1 h. THF wasevaporated off in-vacuo. The residue was adsorbed on silica and purifiedby flash chromatography, eluting with CH₂Cl₂/EtOAc (1:1) to give 78 (38mg, 19%) as a white solid. m/z=395.08 (M+H).

benzhydryl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (79) SU0021-02

(2-Chloroquinolin-3-yl)methanol (119 mg, 0.65 mmol) and 76 (70 mg, 0.35mmol) were dissolved in THF (2 mL). The resulting mixture was stirred atroom temperature for 15 min and then at 80° C. for 1 h. THF wasevaporated off in vacuo. The residue was adsorbed on silica and purifiedby flash chromatography, eluting with CH₂Cl₂/EtOAc (1:1) to give 79 (50mg, 37%) as a white solid. m/z=386.16 (M+H).

benzhydryl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (80) SU0021-02

(4-Methyl-2-phenylpyrimidin-5-yl)methanol (100 mg, 0.50 mmol) and 76(151 mg, 0.75 mmol) were dissolved in THF (2 mL). The resulting mixturewas stirred at room temperature for 15 min and then at 80° C. for 1 h.THF was evaporated off in vacuo. The residue was adsorbed on silica andpurified by flash chromatography, eluting with CH₂Cl₂/EtOAc (1:1) togive 80 (70 mg, 35%) as a white solid. m/z=402.15 (M+H).

(1H-benzo[d]imidazol-2-yl)methyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate(81) VG032-03

(1H-benzo[d]imidazol-2-yl)methanol (200 mg, 1.35 mmol) and 76 (272 mg,1.35 mmol) were dissolved in THF (2 mL). The resulting mixture wasstirred at room temperature for 15 min and then at 80° C. for 1 h. THFwas evaporated off in vacuo. The residue was adsorbed on silica andpurified by flash chromatography, eluting with CH₂Cl₂/EtOAc (1:1) togive 81 (80 mg, 17%) as a white solid. m/z=350.12 (M+H).

(2H-chromen-3-yl)methyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (82)SU033-03

2H-chromene-3-carbaldehyde (500 mg, 3.13 mmol) was dissolved in EtOH (10mL). NaBH₄ (119 mg, 3.13 mmol) was added portionwise at 0° C., withvigorous stirring. The suspension was stirred at 0° C. for 15 min andthen at room temperature for 1.5 h. Solvent was evaporated off in-vacuoto obtain the alcohol intermediate as an oil. This was dissolved in THF(5 mL) and 76 (155 mg, 0.77 mmol) was added. The resulting mixture wasstirred at room temperature for 15 min and then at 80° C. for 1 h. THFwas evaporated off in vacuo. The residue was adsorbed on silica andpurified by flash chromatography, eluting with CH₂Cl₂/EtOAc (1:1) togive 82 (80 mg, 8%) as a white solid. m/z=364.12 (M+H), 727.23 (2M+H).

naphthalen-2-ylmethyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (83)VG037-03

Naphthalen-2-ylmethanol (200 mg, 1.27 mmol) and 76 (279 mg, 1.39 mmol)were dissolved in THF (2 mL). The resulting mixture was stirred at roomtemperature for 15 min and then at 80° C. for 1 h. THF was evaporatedoff in vacuo. The residue was adsorbed on silica and purified by flashchromatography, eluting with CH₂Cl₂/EtOAc (1:1) to give 83 (26 mg, 6%)as a white solid. m/z=360.13 (M+H), 719.25 (2M+H).

benzofuran-2-ylmethyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (84)SU08-03

Benzofuran-2-ylmethanol (300 mg, 2.03 mmol) and 76 (407 mg, 2.03 mmol)were dissolved in THF (2 mL). The resulting mixture was stirred at roomtemperature for 15 min and then at 80° C. for 1 h. THF was evaporatedoff in vacuo. The residue was adsorbed on silica and purified by flashchromatography, eluting with CH₂Cl₂/EtOAc (1:1) to give 84 (130 mg, 18%)as a white solid. m/z=350.09 (M+H), 699.17 (2M+H).

benzo[d]thiazol-2-ylmethyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (85)SU024-3-03

Benzo[d]thiazol-2-ylmethanol (200 mg, 1.21 mmol) and 76 (365 mg, 1.8mmol) were dissolved in THF (2 mL). The resulting mixture was stirred atroom temperature for 15 min and then at 80° C. for 1 h. THF wasevaporated off in vacuo. The residue was adsorbed on silica and purifiedby flash chromatography, eluting with CH₂Cl₂/EtOAc (1:1) to give 85 (90mg, 20%) as a white solid. m/z=367.02 (M+H), 733.13 (2M+H).

4-(furan-2-yl)benzyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (86)SU024-3-03

(4-(Furan-2-yl)phenyl)methanol (100 mg, 0.57 mmol) and 76 (139 mg, 0.69mmol) were dissolved in THF (10 mL). The resulting mixture was stirredat room temperature for 15 min and then at 80° C. for 1 h. THF wasevaporated off in vacuo. The residue was adsorbed on silica and purifiedby flash chromatography, eluting with CH₂Cl₂/EtOAc (1:1) to give thetarget compound (20 mg, 9%) as a white solid. m/z=376.11 (M+H), 751.22(2M+H).

(5-methylbenzo[b]thiophen-2-yl)methyl4-methyl-2-oxo-2H-chromen-7-ylcarbamate (87) SU030-4-03

(5-Methylbenzo[b]thiophen-2-yl)methanol (100 mg, 0.56 mmol) and 76 (136mg, 0.67 mmol) were dissolved in THF (2 mL). The resulting mixture wasstirred at room temperature for 15 min and then at 50° C. for 3 h. THFwas evaporated off in vacuo. The residue was adsorbed on silica andpurified by flash chromatography, eluting with CH₂Cl₂/EtOAc (1:1) togive 87 (38 mg, 18%) as a white solid. m/z=380.09 (M+H), 759.17 (2M+H).

(5-methoxybenzofuran-2-yl)methyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate(88) VG032-05

(5-Methoxybenzofuran-2-yl)methanol 25 (200 mg, 1.12 mmol) and 76 (190mg, 0.95 mmol) were dissolved in THF (10 mL). The resulting mixture wasstirred at room temperature for 15 min and then at room temperature for16 h. THF was evaporated off in vacuo. The residue was adsorbed onsilica and purified by flash chromatography, eluting with hexane/EtOAc(1:1) to give 88 (20 mg, 5%) as a white solid. m/z=380.13 (M+H), 759.26(2M+H).

(5-bromobenzofuran-2-yl)methyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate(89) VG036-05

(5-Bromobenzofuran-2-yl)methanol (100 mg, 0.44 mmol) and 76 (106 mg,0.52 mmol) were dissolved in THF (2 mL). The resulting mixture wasstirred at room temperature for 15 min and then at 80° C. for 1 h. THFwas evaporated off in vacuo. The residue was adsorbed on silica andpurified by flash chromatography, eluting with CH₂Cl₂/EtOAc (1:1) togive 89 (5.0 mg, 3%) as a white solid. m/z=429.10 (M+H).

(5,7-dimethoxybenzofuran-2-yl)methyl4-methyl-2-oxo-2H-chromen-7-ylcarbamate (90) VG041-05

(5,7-dimethoxybenzofuran-2-yl)methanol 5 (50 mg, 0.24 mmol) and7-isocyanato-4-methylcoumarin (58 mg, 0.29 mmol) were dissolved in THF(10 mL). The resulting mixture was stirred at room temperature for 16 h.THE was evaporated off in vacuo. The residue was adsorbed on silica andpurified by flash chromatography, eluting with CH₂Cl₂/EtOAc (1:1) togive 90 (5.0 mg, 3%) as a white solid. m/z=410.04 (M+H).

8. Extended Linkers: Oxybenzyl Ether, Carbamate Benzyl Ether, OxybenzylCarbamate

4-methyl-7-(4-naphthalen-1-ylmethoxy)benzyloxy)-2H-chromen-2-one (91)TLE-M1-SU001B

A suspension of sodium ethoxide (924 mg, 13.6 mmol) in DMF was stirredat 0° C. for 10 min. Ethyl 4-hydroxy benzoate (2.26 g, 13.6 mmol) wasslowly added and resulting mixture was stirred at this temp for 0.5 h,then allowed to reach room temp. To this mixture1-(bromomethyl)naphthalene (2.0 g, 9.0 mmol) was added dropwise[(predissolved in DMF (5 mL)]. Resulting mixture was stirred at roomtemperature for 16 h. DMF was evaporated off in-vacuo and the residuetaken up in EtOAc, and washed with brine, water and 1M NaOH (2×30 mL).The organic layer was dried (MgSO₄) and the solvent evaporated offin-vacuo to yield ethyl 4-(naphthalene-1-ylmethoxy)benzoate (1.7 g, 5.55mmol). This was then dissolved in THF and LiAlH₄ (211 mg, 5.55 mmol) wasadded portionwise, with vigorous stirring. The suspension was stirred atroom temperature for 3 h. THF was evaporated off in-vacuo. The cruderesidue was taken up in EtOAc and washed with water, brine and dried(MgSO₄). EtOAc was evaporated off in-vacuo to obtain(4-(naphthalene-1-ylmethoxy)phenyl)methanol (1.3 g, 4.9 mmol) as a crudeproduct. This was used in the next reaction step without furtherpurification.

(4-(naphthalen-1-ylmethoxy)phenyl)methanol (1.0 g, 3.8 mmol) wasdissolved in toluene (30 mL) and pyridine (305 uL, 3.8 mmol) was added.The solution was cooled to 0° C. PBr₃ (359 uL, 3.8 mmol) was addeddropwise over 15 min. The mixture was brought up to room temperature andstirred for 1 h. It was washed with K₂CO₃ solution and extracted withEtOAc (3×30 mL). The EtOAc layer was washed with brine and dried(MgSO₄). The solvent was evaporated off in-vacuo to obtain1-((4-(bromomethyl)phenoxy)methyl)naphthalene (660 mg, 53%). Thisintermediate was used in the following reaction.

Sodium ethoxide (156 mg, 2.29 mmol) was added to DMF at 0° C., and thesuspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (403 mg,2.29 mmol) was slowly added and resulting mixture was stirred for 0.5 h,and then allowed to reach room temperature. To this mixture1-((4-(bromomethyl)phenoxy)methyl)naphthalene (500 mg, 1.53 mmol) wasadded portionwise. Resulting mixture was stirred at room temperature for16 h. DMF was evaporated off in-vacuo and the residue was taken up inEtOAc, and washed with brine (2×50 mL), water (2×50 mL) and 1M NaOH(2×30 mL). The organic layer was dried (MgSO₄) and purified by flashchromatography, eluting with hexane: EtOAc (2:1) to give 91 (200 mg,31%) as a white solid, Mpt=154-156° C.; H¹ NMR (500 MHz, acetone-d₆):δ=8.10 (1H, d, ArH), 8.00-7.99 (2H, m, Ar), 7.97-7.71 (2H, m, ArH),7.70-7.53 (3H, m, ArH), 7.24 (1H, s, ArH), 7.09 (1H, d, CH), 6.23 (1H,s, CH), 5.68 (2H, s, CH₂), 2.40 (3H, s, CH₃). ¹³C NMR (500 MHz,acetone-d₆, DEPT135): δ=154.6 (qC), 153.4 (qC), 133.3 (qC), 131.1,129.8, 128.9, 128.7, 128.5, 126.9, 126.7, 126.5, 126.4, 126.0, 125.9(11×Ar CH), 125.3, (Ar CH), 123.8 (Ar CH), 114.8 (Ar CH), 113.1 (Ar CH),112.7 (Ar CH), 111.2 (Ar CH), 111.1 (Ar CH), 101.7 (CH), 69.6 & 67.9(2×CH₂), 18.1 (CH₃).

7-(4-(benzhydryloxy)benzyloxy)-4-methyl-2H-chromen-2-one (92)TLE-M1-SU004B

Sodium ethoxide (661 mg, 9.7 mmol) was added to DMF (5 mL) at 0° C.Resulting suspension was stirred for 15 min. Ethyl 4-hydroxy benzoate(1.61 g, 9.7 mmol) was slowly added and resulting mixture was stirred atthis temperature for 0.5 h, then allowed to reach room temperature. Tothis mixture diphenylmethyl bromide (2.0 g, 8.1 mmol) was addedportionwise. Resulting reaction mixture was stirred at room temperaturefor 16 h. DMF was evaporated off in-vacuo and the residue was taken upin EtOAc, and washed with brine, water and 1M NaOH (2×30 mL). Theorganic layer was dried (MgSO₄) and the solvent evaporated off in-vacuoto yield ethyl 4-(benzhydryloxy)benzoate (1.0 g, 3.0 mmol). This wasthen dissolved in THF (5 mL) and LiAlH₄ (114 mg, 3.0 mmol) was addedportionwise, with vigorous stirring. The suspension was stirred at roomtemperature for 3 h. THE was evaporated off in-vacuo. The crude residuewas taken up in EtOAc and washed with water, brine and dried (MgSO₄).EtOAc was evaporated off in-vacuo to obtain(4-(benzhydryloxy)phenyl)methanol (760 mg, 2.62 mmol) as a crudeproduct. This was used in the next reaction step without furtherpurification.

(4-(Benzhydryloxy)phenyl)methanol (500 mg, 1.72 mmol) was dissolved intoluene (20 ml) and pyridine (139 uL, 1.72 mmol) was added. The solutionwas cooled to 0° C. PBr₃ (163 uL, 1.72 mmol) was added dropwise over 15min. The resulting mixture was stirred at room temperature for 1 h. Itwas then washed with saturated K₂CO₃ solution and extracted with EtOAc(3×30 mL). The EtOAc layer was washed with brine and dried (MgSO₄). Thesolvent was evaporated off in-vacuo to obtain((4-(bromomethyl)phenoxy)methylene)dibenzene as an oil, (450 mg, 74%).This intermediate was used in the following reaction.

Sodium ethoxide (87 mg, 1.28 mmol) was added to DMF (3 mL) at 0° C., andthe suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (225mg, 1.28 mmol) was slowly added and resulting mixture was stirred atthis temperature for 0.5 h, then allowed to reach room temperature. Tothis mixture ((4-(bromomethyl)-phenoxy) methylene)dibenzene (300 mg,1.53 mmol) was added portionwise. Resulting mixture was stirred at roomtemperature for 16 h. DMF was evaporated off in-vacuo and the residuewas taken up in EtOAc, and washed with brine (2×50 mL), water (2×50 mL)and 1M NaOH (2×30 mL). The organic layer was dried (MgSO₄) and purifiedby flash chromatography, eluting with hexane: EtOAc (2:1) to give 92 (80mg, 21%) as a white solid. Mpt=179-181° C. H¹ NMR (500 MHz, acetone-d₆):δ=7.66 (1H, d, ArH), 7.56 (4H, d, ArH), 7.39-7.34 (6H, m, ArH), 7.08(2H, d, ArH), 6.97 (2H, d, ArH), 6.93 (2H, d, ArH), 6.51 (1H, s, CH),6.12 (1H, s, CH), 5.12 (2H, s, CH₂), 2.42 (3H, s, CH₃).

7-(4-(benzofuran-2-ylmethoxy)benzyloxy)-4-methyl-2H-chromen-2-one (94)SU010B-02

Ethyl 4-(benzofuran-2-ylmethoxy)benzoate (93)

Sodium ethoxide (580 mg, 8.5 mmol) was added to DMF (10 mL) at 0° C.Resulting suspension was stirred for 15 min. Ethyl 4-hydroxy benzoate(1.4 g, 8.5 mmol) was slowly added and resulting mixture was stirred atthis temperature for 0.5 h, then allowed to reach room temp. To thismixture 10 (1.5 g, 7.1 mmol) was added dropwise, predissolved in DMF (5mL). Resulting reaction mixture was stirred at room temperature for 2 h.DMF was evaporated off in-vacuo and the residue was taken up in EtOAc,and washed with brine, water and 1M NaOH (2×30 mL). The organic layerwas dried (MgSO₄) and the solvent evaporated off in-vacuo to give 93 asa white solid (920 mg, 44%). m/z=423.18 (M+H). H¹ NMR (500 MHz,Acetone-d₆): δ=8.0 (2H, d, ArH), 7.60 (1H, d, ArH), 7.30-7.15 (2H, m,ArH), 7.27-7.19 (1H, m, CH), 7.00 (2H, d, ArH), 6.80 (1H, s, CH), 5.20(2H, s, CH₂), 4.10 (2H, q, CH₂), 1.25 (3H, t, CH₃).

7-(4-(benzofuran-2-ylmethoxy)benzyloxy)-4-methyl-2H-chromen-2-one (94)U010B-02

Compound 93 (400 mg, 1.29 mmol) was dissolved in THF (15 mL) and LiAlH₄(49 mg, 1.29 mmol) was added portionwise, with vigorous stirring. Thesuspension was stirred at room temperature for 1 h. THE was evaporatedoff in-vacuo. The crude residue was taken up in EtOAc and washed withwater, brine and dried (MgSO₄). EtOAc was evaporated off in-vacuo toobtain (4-(benzofuran-2-ylmethoxy)phenyl)methanol (220 mg, 67%) as acrude product. This was dissolved in toluene (10 mL). The solution wascooled to 0° C. PBr₃ (98 μL, 1.04 mmol) was added dropwise over 15 min.The resulting mixture was stirred at room temperature for 1 h. It wasthen washed with saturated K₂CO₃ solution and extracted with EtOAc (3×30mL). The EtOAc layer was washed with brine and dried (MgSO₄). Thesolvent was evaporated off in-vacuo to obtain2-((4-(bromomethyl)phenoxy)methyl)benzofuran as a colourless oil, (132mg). This intermediate was used in the following reaction.

Sodium ethoxide (43 mg, 0.63 mmol) was added to DMF at 0° C., and thesuspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (110 mg,0.63 mmol) was slowly added and resulting mixture was stirred at thistemperature for 0.5 h, then allowed to reach room temperature. To thismixture 93 (132 mg, 0.42 mmol) was added portionwise. Resulting reactionmixture was stirred at room temp for 16 h. DMF was evaporated offin-vacuo and the residue was taken up in EtOAc, and washed with brine(2×50 mL), water (2×50 mL) and 1M NaOH (2×30 mL). The organic layer wasdried (MgSO₄) and purified by flash chromatography, eluting with hexane:EtOAc (2:1) to give 94 (80 mg, 47%) as a white solid. Mpt=153-155° C.m/z=413 (M+H). H¹ NMR (500 MHz, Acetone-d₆): δ=7.56-7.46 (2H, m, CH),7.40 (1H, t, J=8.2, CH), 7.34 (2H, d, J=8.50, CH), 7.27-7.19 (1H, m,CH), 7.14-7.09 (1H, m, CH), 7.00-6.98 (2H, d, CH, J=11.8), 6.86-6.80(3H, m, CH), 5.99 (1H, s, CH), 5.14 (2H, s, CH₂), 5.03 (2H, s, CH₂),2.28 (3H, s, CH₃).

naphthalen-1-ylmethy 4-((4-methy-2-oxo-2H-chromen-7yloxy)methyl)phenylcarbamate (95) VG021-03

To a stirred solution of naphthalene methanol (4.0 g, 25.3 mmol) in THF(30 mL) was added TEA (100 uL). To this was added dropwise,ethylcyanobenzoate (4.0 g, 21.0 mmol), predissolved in THF (10 mL). Theresulting solution was stirred at room temperature for 16 h. Solvent wasevaporated off to give a crude intermediate, ethyl4-((naphthalen-1-ylmethoxy)carbonylamino)benzoate (1.3 g). This was thendissolved in THF (15 mL) and LiAlH₄ (141 mg, 3.75 mmol) was addedportionwise, with vigorous stirring. The suspension was stirred at roomtemperature for 1 h. THF was evaporated off in-vacuo. The crude residuewas taken up in EtOAc and washed with water, brine and dried (MgSO₄).EtOAc was evaporated off in-vacuo to obtain (naphthalen-1-ylmethyl4-(hydroxymethyl)phenylcarbamate (500 mg, 1.62 mmol), as a crudeproduct. This was dissolved in toluene (10 mL). The solution was cooledto 0° C. PBr₃ (154 uL, 1.62 mmol) was added dropwise over 15 min. Theresulting mixture was stirred at room temperature for 1 h. The solventwas evaporated off in-vacuo to obtain naphthalen-1-ylmethyl4-(bromomethyl)phenylcarbamate as an oil, (300 mg). This intermediatewas used in the following reaction without further purification.

Sodium ethoxide (44 mg, 0.65 mmol) was added to DMF at 0° C., and thesuspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (114 mg,0.70 mmol) was slowly added and the mixture was stirred at thistemperature for 0.5 h, then allowed to reach room temperature. To thismixture 2-((4-(bromomethyl)phenoxy)methyl)benzofuran (200 mg, 0.42 mmol)was added portionwise. Resulting mixture was stirred at room temperaturefor 16 h. DMF was evaporated off in-vacuo and the residue was taken upin EtOAc, and washed with brine (2×50 mL), water (2×50 mL) and 1M NaOH(2×30 mL). The organic layer was dried (MgSO₄) and purified by flashchromatography, eluting with hexane: EtOAc (2:1) to give 95 (160 mg,63%) as a white solid; Mpt=153-155° C. m/z=488.2 (M+H). H¹ NMR (500 MHz,Acetone-d₆): δ=7.56-7.46 (2H, m, CH), 7.40 (1H, t, J=8.2, CH), 7.34 (2H,d, J=8.50, CH), 7.27-7.19 (1H, m, CH), 7.14-7.09 (1H, m, CH), 7.00-6.98(2H, d, CH, J=11.8), 6.86-6.80 (3H, m, CH), 5.99 (1H, s, CH), 5.14 (2H,s, CH₂), 5.03 (2H, s, CH₂), 2.28 (3H, s, CH₃).

4-(benzofuran-2-ylmethoxy)benzyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate(96) SU024-1-03)

Compound 93 (300 mg, 1.01 mmol) was dissolved in THF (15 mL) and LiAlH₄(38 mg, 1.01 mmol) was added portionwise, with vigorous stirring. Thesuspension was stirred at room temperature for 1 h. THF was evaporatedoff in-vacuo. The crude residue was taken up in EtOAc and washed withwater, brine and dried (MgSO₄). EtOAc was evaporated off in-vacuo toobtain (4-(benzofuran-2-ylmethoxy)phenyl)methanol (150 mg, 0.59 mmol),as a crude product. This was used in the next step without furtherpurification.

Sodium ethoxide (40 mg, 0.59 mmol) was added to DMF at 0° C., and thesuspension was stirred for 10 min.(4-(benzofuran-2-ylmethoxy)phenyl)methanol (150 mg, 0.59 mmol), wasadded and resulting mixture was stirred at this temperature for 0.5 h,then allowed to reach room temperature. To this mixture 76 (130 mg, 0.65mmol) was added portionwise. Resulting mixture was stirred at roomtemperature for 2 h. DMF was evaporated off in-vacuo. The crude residuewas purified by flash chromatography, eluting with hexane: EtOAc (2:1)to give the target compound (20 mg, 8%) as a white solid. m/z=456.10(M+H). H¹ NMR (500 MHz, DMSO-d₆): δ=10.22 (1H, bs, NH), 7.69-7.64 (2H,m, ArH), 7.58 (1H, d, J=8.15 Hz, ArH), 7.55 (1H, s, ArH), 7.42 (2H, d,J=8.00 Hz, ArH), 7.33 (1H, t, J=7.70 Hz, ArH), 7.26 (1H, t, J=7.5 Hz,ArH), 7.11 (2H, d, J=8.28 Hz, ArH), 7.06 (1H, s, ArH), 6.23 (1H, s,ArH), 5.29 (2H, s, CH₂), 5.12 (2H, s, CH₂), 2.37 (3H, s, CH₃).

7-(4-((5-methoxybenzofuran-2-yl)methoxy)benzyloxy)-4-methyl-2H-chromen-2-one(97) VG040-05

Sodium ethoxide (62 mg, 0.90 mmol) was added to DMF (3 mL) at 0° C.Resulting suspension was stirred for 15 min. Ethyl 4-hydroxy benzoate(148 mg, 0.90 mmol) was slowly added and resulting mixture was stirredat this temperature for 0.5 h, then allowed to reach room temperature.To this mixture 26 (180 mg, 0.75 mmol) was added. Resulting reactionmixture was stirred at room temperature for 1 h. DMF was evaporated offin-vacuo to give a crude intermediate (150 mg). This was then dissolvedin THF (5 mL) and LiAlH₄ (34 mg, 0.90 mmol) was added portionwise, withvigorous stirring. The suspension was stirred at room temperature for 1h. THF was evaporated off in-vacuo. The crude residue was taken up inEtOAc and washed with water, brine and dried (MgSO₄). EtOAc wasevaporated off in-vacuo to obtain(4-((5-methoxybenzofuran-2-yl)methoxy)phenyl)methanol (120 mg) as acrude product. This was dissolved in toluene (4 mL). The solution wascooled to 0° C. PBr₃ (84 μL, 1.04 mmol) was added dropwise over 5 min.The resulting mixture was stirred at room temperature for 1 h. Thesolvent was evaporated off in-vacuo to obtain2-((4-(bromomethyl)phenoxy)methyl)-5-methoxybenzofuran as a colourlessoil, (90 mg). This intermediate was used in the following reaction.

Sodium ethoxide (22 mg, 0.31 mmol) was added to DMF (2 mL) at 0° C., andthe suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (54mg, 0.31 mmol) was slowly added and resulting mixture was stirred atthis temperature for 0.5 h. To this mixture2-((4-(bromomethyl)phenoxy)methyl)-5-methoxybenzofuran (90 mg, 0.23mmol) was added portionwise. Resulting reaction mixture was stirred atroom temp for 16 h. DMF was evaporated off in-vacuo and the residue waspurified by flash chromatography, eluting with hexane: EtOAc (1:1) togive 94 (22 mg, 7%) as a white solid. m/z=443 (M+H).

4-(benzhydryloxy)benzyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (98)SU032-02

Sodium ethoxide (300 mg, 4.05 mmol) was added to DMF (10 mL) at 0° C.Resulting suspension was stirred for 10 min. Ethyl 4-hydroxy benzoate(739 mg, 4.05 mmol) was slowly added and resulting mixture was stirredat this temperature for 20 min. To this mixture diphenylmethyl bromide(1.0 g, 4.05 mmol) was added portionwise. Resulting reaction mixture wasstirred at room temperature for 16 h. DMF was evaporated off in-vacuoand the residue was taken up in EtOAc, and washed with water and brine.The organic layer was dried (MgSO₄) and the solvent evaporated offin-vacuo to yield ethyl 4-(benzhydryloxy)benzoate (830 mg). This wasthen dissolved in THF (5 mL) and LiAlH₄ (114 mg, 3.0 mmol) was addedportionwise, with vigorous stirring. The suspension was stirred at roomtemperature for 3 h. THF was evaporated off in-vacuo. The crude residuewas taken up in EtOAc and washed with water, brine and dried (MgSO₄).EtOAc was evaporated off in-vacuo to obtain(4-(benzhydryloxy)phenyl)methanol (760 mg, 2.62 mmol) as a crudeproduct. This was used in the next reaction step without furtherpurification.

A solution of (4-(Benzhydryloxy)phenyl)methanol (100 mg, 0.34 mmol) and76 in toluene (10 ml). was refluxed for 2 h. The reaction was allowed tocool to room temperature and the resulting precipitate formed wasfiltered and washed with cold ether and EtOAc to give 98 (100 mg, 60%)as a white solid. m/z=491.55 (M+H).

Biological Activity Example 1: CYP1B1 Metabolism of Prodrugs SubstituentEffect on the Fragmentation of Benzofuran Ether and Carbamate LinkedCoumarins by CYP1 Isoenzymes and Human Liver Microsomes (HLM).

Commercially available Supersomal™ CYP1A1, CYP1A2, CYP1B1, and pooledhuman liver microsomes (supplied BD Gentest, Oxford, UK) comprised anenzymatic screen to identify structure activity relationships (SARs)underlying the structural features which control the efficiency andselectivity of prodrug fragmentation by CYP1B1 expressed in cancerrelative to cytochrome P450 enzymes expressed in normal tissuesincluding the liver. HLMs are derived from human patient liver andaccording to the supplier contain a battery of cytochrome P450sincluding CYP1A2, CYP2A6, CYP2B6, CYP2B6, CYP2C8, CYP2C9, CYP2D6,CYP2E1, CYP3A4, and CYP4A but not CYP1A1 or CYP1B1.

Typical Supersomal™ CYP1A1, CYP1A2, CYP1B1 enzyme metabolism studiesused 10 pmol enzyme, 100 μmol dm⁻³ NADPH, in 10 mmol dm⁻³ potassiumphosphate buffer at pH 7.4 and 37° C. Supersomal™ enzyme metabolism wasstarted by adding a stock solution of prodrug dissolved in DMSO to givea final concentration of 10 μmol dm⁻³ prodrug and 0.5% DMSO. HLMscreening used 60 microlitre microsomes, 100 μmol dm⁻³ NADPH, in 10 mmoldm⁻³ potassium phosphate buffer at pH 7.4 and 37° C., in 1.5 ml totalreaction volume.

Compounds of the invention comprise a series of heteroaromatic triggerscoupled to ether and carbamate linkers to the hydroxyl group of7-hydroxy-4-methycoumarin and 7-amino-4-methylcoumarin, respectively.Further examples of the invention comprise compounds were heteroaromatictriggers are coupled via the so-called extended oxybenzyl ether linker(—Ar—CH(Z⁷)X³—=-phenyl-CH₂O—) to the hydroxyl group of7-hydroxy-4-methycoumarin. Further examples of the invention comprisecompounds where heteroaromatic triggers are coupled via the so-calledextended oxybenzyl carbamate linker to the amino group of7-amino-4-methycoumarin. Further examples of the invention comprisecompounds where heteroaromatic triggers are coupled via a carbamatebenzyl ether linker to the hydroxyl group of 7-hydroxy-4-methycoumarin.

Both 7-hydroxy-4-methycoumarin and 7-amino-4-methylcoumarin arepartially deprotonated at physiological pH 7.4 and both coumarin anionsare highly fluorescent with fluorescence emission wavelength maxima of450 and 445 nm, respectively. When the coumarins are coupled tohetero-aromatic triggers via linkers described in this invention thefluorescence of the coumarin anion is quenched. Therefore, enzymatichydroxylation of the hetero-aromatic trigger and resultant linkerfragmentation can be monitored in real time by release of the coumarinanion by kinetic fluorimetry. This prodrug design strategy has beensuccessfully used to monitor the fragmentation of so-called bioreductivehypoxia-activated prodrugs by P450 reductase not to be confused withCYP1B1 which is a mono-oxygenase enzyme (See, e.g.: Everett S A et al.,Modifying rates of reductive elimination of leaving groups fromindolequinone prodrugs: a key factor in controlling hypoxia-selectivedrug release”, Biochem Pharmacol., 63: 1629-39, 2002).

Release of the coumarin anion indicative of linker fragmentation wasmonitored using a 1 cm path length fluorescence cell in a Cary Eclipsekinetic flourimeter with excitation and emission slits set at 5 nm.Coumarin anion release from compounds of the invention was detected atthe excitation wavelength λ_(ex)=350-nm and the emission wavelengthλ_(em)=450 nm. Change in fluorescence intensity was quantified against alinear calibration plot of fluorescence intensity versus coumarinconcentration (0 to 3.5 μmol dm⁻³) in 10 mmol dm⁻³ potassium phosphatebuffer at pH 7.4 using the same instrument settings as for enzymemetabolism.

Specific fragmentation activities (in pmol coumarin min⁻¹ pmolcytochrome P450⁻¹) for the CYP1 isoenzyme and HLM-activatedfragmentation and release of coumarin from benzofuran ether andcarbamate-linked coumarins are shown in Table 3. An electron donatingsubstituent (Me, MeO) or electron withdrawing substituents (Cl, Br, F)in one or both of the 5- and 7-position of the benzofuran has asignificant effect on both fragmentation specificity and efficiency. The4- and 6-positions on the benzofuran are left unsubstituted (R⁴ andR⁶═H) as they are likely positions for enzymatic hydroxylation necessaryto induce ether or carbamate linker fragmentation according to theproposed mechanism. The structure activity relationship (SAR) governingthe substituent effect at the 5- and 7-position on the benzofuran andCYP1 isoenzyme-induced fragmentation efficiency and selectivity are notpredictable for either ether or carbamate linker fragmentation.

SU10A (see Table 3 below) where Z³═H, Z⁵═H bearing an ether-linkedcoumarin is fragmented by CYP1A1, CYP1A2, and CYP1B1 as well as HLM. ForHLM the inclusion of 10 μmol dm⁻³ α-naphthoflavone (a CYP1-selectiveenzyme inhibitor) inhibits SU10A fragmentation indicating that CYP1A2 issolely responsible for HLM-mediated coumarin release. Benzofuran istherefore a generic trigger moiety that can facilitate the fragmentationof ether-linked prodrugs by CYP1 isoenzymes.

Electron withdrawing substituents on the benzofuran in VG016-04 (seealso Table 3 below) where Z³═F, Z⁵═F inhibits CYP1 isoenzyme andHLM-induced fragmentation of the ether linker. However, electrondonating substituents in VG035-05 (see also Table 3 below) where Z³=MeO,Z⁵=MeO results in CYP1B1-specific fragmentation of the ether bond as nolinker fragmentation is observed for CYP1A or HLM. The specificfragmentation activity for VG035-05 with CYP1B1 is 13.65±1.00 pmolcoumarin min⁻¹ pmol cytochrome P450⁻¹ is the highest efficiency of thevarious benzofuran ether-linked coumarins investigated (see Table 3below). The 5,7-dimethoxybenzofuran moiety can therefore be used tospecifically trigger the fragmentation of ether-linked prodrugs byCYP1B1 an enzyme which is over-expressed in cancer.

In marked contrast to VG035-04 (which contains an ether-linkedcoumarin), VG041-05 (which contains the corresponding carbamate-linkedcoumarin) where Z³=MeO, Z⁵=MeO is selectively fragmented by CYP1A1 (butnot CYP1A2 or CYP1B1) with a specific fragmentation activity=5.51±0.06pmol coumarin min⁻¹ pmol cytochrome P450⁻¹. According to Table 3 belowall compound examples of structure B containing a carbamate linker arefragmented by CYP1A1 but not CYP1B1. The only exception is VG032-05where X³=MeO, Z⁵=MeO giving a CYP1B1 specific fragmentationactivity=1.53±0.09 pmol coumarin min⁻¹ pmol cytochrome P450⁻¹, which is˜6-fold lower than VG027-05 which contains an ether linker.

Example 2: Combining the Model Prodrug Library with a CYP1B1 SubstratePrediction Model Links Substrate Specificity to Prodrug Activation andFragmentation Performing a High-Throughput Screen (HTS) to Build aBioactivity Dataset for CYP181

The target enzyme CYP1B1 was screened against two commercial librariesincluding the ChemDiv Diversity 50,000 test compound collection and theChemDiv Kinase Targeted 10,000 test compound collection with a view toidentifying activity differentiating substructures and a largebioactivity dataset from which to build a substrate specificity model.The HTS was performed in miniaturized 384-well format using a liquidhandler (Beckman FXp), bulk dispensers (Matrix Wellmate) and aluminescent plate reader (Molecular Devices Analyst AD plate Reader).P450-Glo™ Assays provide a luminescent method for measuring cytochromeP450 activity. A conventional reaction is performed by incubating thehuman supersomal CYP1B1 plus reductase (BD Gentest™, UK) recombinantenzyme with a luminogenic cytochrome P450 substrate, namely Luciferin 6′chloroethyl ether (Luciferin-CEE) which is a substrate for CYP181 butnot for luciferase. Luciferin-CEE is converted to a luciferin productthat is detected in a second reaction with Luciferin Detection Reagent(CYP1B1 Luminescent Assay Kit, P450-Glo™ from Promega, Madison, USA).The reagent simultaneously stops the cytochrome P450 reaction andinitiates a stable luminsescent signal with a half-life >2 h. The amountof light produced in the second reaction is proportional to the activityof CYP1B1. The biochemical end-point was substrate inhibition of theenzyme (0.5 pmol/well) working at the apparent K_(m) for Luciferin-CEE(20 μmol dm⁻³). The assay is characterized by excellent Z′-factorstypically greater than 0.6 (where a Z′=1.0 denotes a perfectly robusthighly reproducible assay) when run in 384-well format. The negativecontrol was the level of activity which defined the unmodified state ofthe enzyme target while the positive control was the level of activitywhich defined a hit. The negative control contained theCYP1B1/KPO₄/NADPH/substrate reaction mixture and an equivalentconcentration of 1% DMSO used for solubilization of the test compounds.The positive control in the assay contained the CYP1B1/KPO₄/NADPHsubstrate reaction mixture with α-naphthoflavone which completelyinhibits CYP1B1 enzyme activity at a final concentration of 5 μmol dm⁻³.The positive and negative controls were deposited in the outer columnsof every 384-well plate with the test compounds deposited in theremaining 320 wells. The definition of a hit is a test compound that isa substrate inhibitor CYP1B1 activity by 80-100% at a concentration of0.5 μmol dm⁻³.

Pipeline Pilot (Scitegic, San Diego, USA) was used to streamline andintegrate the large quantity of data to identify SARs from the CYP1B1HTS, supported by computational scientists at the UCSF SMDC. Thesoftware was used to identify (1) preliminary SAR results of hits versusnon-hits, (2) determine physicochemical properties e.g. molecularweight, calculated log P, H-atom donor/acceptor interactions of the hitpopulation, (3) determine the frequency of ring fragments and functionalgroups, and (4) define an in silico model for the prediction of CYP1B1substrate inhibition as a basis for future prodrug design. Importantly,s significant number of the hits˜10% had a molecular weight between400-500, the latter being the maximum molecular weight of test compoundsavailable in both compound collections. This information defined themaximum molecular weight of the prodrug permissible whilst maintainingCYP1B1 substrate specificity. Structural analysis of hit scaffolds inSARvision v2 from CHEMAPPS™ (La Jolla, Calif., USA) confirmed that testcompounds did not support the correct functional group (e.g. a triggerhydroxymethyl substituent) for direct integration into the couplingchemistry reviewed in scheme 1. However, by identifying ring fragmentsof high frequency in hits versus non-hits it was possible to identifytemplates for trigger moieties which could then be functionalizedappropriately for coupling reactions.

An in Silico Model for Predicting Cytochrome P450 Substrate Inhibitionin Support of Prodrug Design.

A major challenge in prodrug design is to define the strategy tointegrate the trigger, linker and effector chemistry whilst maintainingsubstrate specificity for the target enzyme. The CYP1B1 HTS wasextremely valuable in identifying potential trigger moieties butsubsequent ‘hit to lead’ chemistry incorporating a linker and effectordrug could mean that the final prodrug structure would not be optimalfor target enzyme activation. Optimal usage of the vast amount ofstructural data from the two HTS screens (totaling 60,000 testcompounds) was achieved by developing an in silico prediction model ofcytochrome P450 1B1 substrate inhibition using Gaussian Kernel weightedk-nearest neighbour (k-NN) algorithm based on Tanimoto similaritysearches on extended connectivity fingerprints. The optimal parametersof the CYP1B1 kernel weighted k-NN model were chosen using leave-one-outcross validation on a training set selected from 45,000 and 9,000 testcompounds from the ChemDiv Diverse and Kinase libraries. The remainderof the test compounds, 6,000 in total, were used as an internal test setto confirm the accuracy of the model to predict substrate inhibition.Any test compounds exhibiting >20 but <80% inhibition were designatednon-classified. The model accurately predicted 89% of the classifiednon-substrate inhibitors and 95% of the classified substrate inhibitors.CYP1B1 substrate prediction model protocol was uploaded into theScitegic Web Port to facilitate the docking of putative prodrugstructures through an interface to standard chemistry drawing packagessuch as ChemDraw/IsisDraw.

Validation of the CYP1B1 Substrate Prediction Model Using an ExternalTest Set of Compounds

A 384-well stock plate constituting an external test set for the CYP1B1substrate prediction model was constructed and included: (1) knownCYP1B1 substrate inhibitors including, for example,tetramethoxystilbene, β-estradiol, α-napthoflavone, ethoxyresorufin,resveratrol, (2) compounds which are not CYP1B1 substrate inhibitorsincluding, for example, quinidine (a potent specific inhibitor ofCYP2D6), sulfaphenazole (a potent specific inhibitor of CYP2C9), and (3)the model prodrugs VG016-05 and VG035-05, and (4) and thephosphoramidate mustard prodrugs SU025-04 and SU046-04. The externaltest set stock concentration was 10 mmol dm⁻³ in DMSO and the percentageCYP1B1 substrate inhibition at a final concentration of 0.5 mmol dm⁻³was determined using the same methods described for the main CYP1B1 HTS.Experiments were perfomed in triplicate to give a mean % substrateinhibition of CYP1B1 activity±standard deviation. All the external testset structures were submitted as queries to the CYP1B1 substrateprediction model via the Scitegic Web Port to generate predicted valuesof % substrate inhibition of CYP1B1 in order to compare with the actualbiochemical measurement of % substrate inhibition.

The comparative actual and predicted % substrate inhibition for CYP1B1values were as follows:

% CYP1B1 substrate inhibition compound actual predicted accuracytetramethoxystilbene 95.76 ± 0.33 97.34 98% β-estradiol, 72.34 ± 0.4576.12 95% α-napthoflavone 99.12 ± 0.23 92.45 93% ethoxyresorufin 92.13 ±0.56 87.23 95% resveratrol 72.34 ± 0.56 76.45 95% sulfaphenazole  2.89 ±0.15 4.25 68% quinidine  1.63 ± 0.34 2.56 64% VG016-05  2.45 ± 0.32 2.9882% VG035-05 97.34 ± 0.32 93.56 96% SU025-04 91.22 ± 0.48 87.36 96%SU046-04 96.45 ± 0.22 92.34 96%

The CYP1B1 substrate prediction model was accurate in predicting %substrate inhibition of CYP1B1 across multiple classes of compound witha broad range of activity confirming validation of the model using anexternal test set of compounds. Importantly, in terms of an inventivestep the model was accurately able to predict the activity of the twomodel prodrugs VG016-05 and VG035-05 in terms of CYP1B1 substrateinhibition which can be linked directly to the efficiency of prodrugfragmentation and release of the 7-hydroxy-4-methy coumarin anion.According to Table 3 electron donating or electron withdrawingsubstituents in the R⁵ and R⁷ of the benzofuran trigger activate thesemodel prodrugs to aromatic hydroxylation and fragmentation. When R⁵ andR⁷═F, i.e. electron withdrawing substituents as in VG016-05 the modelprodrug is not activated by CYP1B1 as accurately predicted and as aconsequence there is no fragmentation of the linker. In marked contrastwhen R⁵ and R⁷=MeO, i.e. electron donating substituents as in VG035-05the model prodrug is activated by CYP1B1 as accurately predictedresulting in fragmentation of the linker with high efficiency.Incorporation of the dimethoxybenzofuran trigger moiety into thephosphoramidate mustard prodrugs SU025-04 and SU046-04 generate compoundwhich are accurately predicted to be good substrate inhibitors ofCYP111. In conclusion, the combination of model prodrug libraries andCYP1B1 substrate prediction models based on a database of CYP1B1bioactivity facilitate the design of specific CYP1B1-activated prodrugs.

Example 3: Prodrug Cytotoxicity in Wild-Type CHO Cells and CHO CellsEngineered to Express CYP1A1 and CYP1B1 Isozymes

Engineered CHO cells were used to demonstrate selective cell killingmediated by CYP1 expression. In the experiments described below,compounds were exposed to wild-type CHO cells engineered to expresseither CYP1A1 (CHO/CYP1A1) or CYP1B1 (CHO/CYP1B1) enzymes.

CHO cells: Chinese Hamster Ovary (CHO) DUKXB11 cells were grown understandard cell culture conditions in α-MEM supplemented with 10% FCS, 1unit/ml each of hypoxanthine and thymidine, and penicillin (100 IU/ml)and streptomycin (100 μg/ml) according to literature methods (Ding S, etal., Arch. Biochem. Biophys., 348: 403-410, 1997, the contents of whichare incorporated herein by reference). Cells were grown at 37° C. in ahumidified atmosphere plus 5% CO₂.

CHO/CYP1A1 and CHO/CYP1B1 cells: CHO cells containing recombinant CYP1A1and recombinant CYP1B1 co-expressing P450 reductase, namely (CHO/CYP1A1)and (CHO/CYP1B1) respectively, were cultured using the standard culturemedium for CHO cells supplemented with 0.4 mg/ml G418 disulfate salt and0.3 μM methotrexate (Sigma/Aldrich Co., Gillingham, Dorset, UK)according to methods described in the literature (ibid.) Cells weregrown at 37° C. in a humidified atmosphere plus 5% CO₂.

Recombinant CYP1A1 and CYP1B1 Expression

Dihydrofolate reductase (DHFR) gene amplification of either human cDNACYP1A1 or cDNA CYP1B1 in CHO cells was used to achieve high levels offunctional enzyme when co-expressed with human P450 reductase (ibid.;Ding S, et al., Biochem J., 356(Pt 2): 613-9, 2001). Modified CYP1A1 orCYP1B1 cDNA was digested and ligated into to the mammalian expressionvector pDHFR to generate the plasmids pDHFR/1A1 and pDHFR/1B1,respectively (ibid.) Cell culture and DNA transfection into CHO DUKXB11was carried out according to methods described in the literature andtransfected cells selected for the DHFR+ phenotype by growth innucleoside deficient medium (ibid.) DHFR+ clones were pooled, and grownon increasing concentrations of MTX (0.02 to 0.1 μM) for amplificationof transfected CYP1A1 or CYP1B1 cDNA. Cell clones that survived 0.1 mMMTX selection were isolated then further selected with 0.3 μM MTX. Theresulting cell lines were analysed for CYP1A1 or CYP1B1 expression byimmunoblotting. Cell lines expressing a high level of each enzyme werestably transfected with plasmid pcDNA/HR containing a full length humancytochrome P450 reductase (CPR) cDNA, and selected with G418 (0.8 mg/ml)and MTX (0.3 μM) according to methods described in the literature(ibid.) After isolation of resistant clones the concentration of G418was reduced to 0.4 mg/ml and the homogeneity of the cell lines assuredby repeated cloning. The CHO cell line transfected with the plasmidcarrying cDNA CYP1A1 subsequently transfected with CPR cDNA wasdesignated CHO/CYP1A1 and the CHO cell line transfected with the plasmidcarrying cDNA CYP1B1 subsequently transfected with CPR cDNA wasdesignated CHO/CYP1B1.

Immunochemical Detection of CYP1A1 and CYP1B1

Cells were harvested and lysed by sonication using standard methods inthe literature (Ding S, et al., 1997, the contents of which areincorporated herein by reference). Proteins (typically 50 μg of lysate)were separated by SDS/PAGE, transferred to a nitrocellulose membrane andprobed using standard methods (Paine M J, et al., Arch. Biochem.Biophys., 328: 380-388, 1996, the contents of which are alsoincorporated herein by reference). Human CYP1A1 plus reductaseSupersomes™, human CYP1A2 plus reductase Supersomes™ and CYP1B1 plusreductase Supersomes™ (BD Biosciences, Oxford, UK) were used as positivecontrols (typically 0.03 to 0.3 pmole) for immunochemical detection ofenzyme expression in cell lines. A WB-1B1 primary antibody (dilution1:1500, BD Biosciences, Oxford, UK) and an anti-CYP1A2 antibody whichcross reacts with CYP1A1 (dilution 1:2000, Cancer Research Technology,London, UK) were used to detect CYP1B1 and CYP1A1 expression,respectively. The secondary antibody was goat anti-rabbit IgG used at a1:500 dilution. Immunoblots were developed using the EnhancedChemiluminescence (ECL) Western-blot detection kit (GE Healthcare LifeSciences, Amersham, Buckinghamshire, UK).

Western-Blot Characterization of CYP1A1 and CYP1B1 Expression inEngineered CHO Cells

FIG. 1a of the accompanying drawings is a typical western-blot showingthe detection of CYP1B1 protein expression in lysate from the CHO/CYP1B1cell line which is detectable in neither the untransfected CHO DUKXB11cells nor the CHO/CYP1A1 cell line. The band corresponds to a molecularweight of 56 kDa and matches the band for human CYP1B1 Supersomal™enzyme. FIG. 1b is a typical western-blot showing the detection ofCYP1A1 protein expression in lysate from the CHO/CYP1A1 cell line whichis detectable in neither the untransfected CHO DUKXB11 cells nor theCHO/CYP1B1 cell line. The band corresponds to a molecular weight of 60kDa and matches the band for human CYP1A1 Supersomal™ enzyme detected bythe cross reactivity of the anti-CYP1A2 antibody.

Functional CYP1 Enzyme Activity

The ethoxyresorufin O-deethylation (EROD) assay is widely used toconfirm functional CYP1 activity (Chang T K and Waxman D J, “EnzymaticAnalysis of cDNA-Expressed Human CYP1A1, CYP1A2, and CYP1B1 with7-Ethoxyresorufin as Substrate”, Methods Mol. Biol., 320: 85-90, 2006,the contents of which are incorporated herein by reference). The assaydetermines O-dealkylation of 7-ethoxyresorufin by CYP1A1, CYP1A2, andCYP1B1 to generate the enzymatic product resorufin, which is monitoredcontinuously by fluorescence emission at 580 nm. An alternative assayfor measuring enzyme activity is the commercially available PromegaP450-Glo™ Assay utilizing Luciferin-CEE as a luminogenic substrate forCYP1 enzymes in Cali J J, et al., Expert. Opin. Drug MetabolismToxicol., 2(4): 629-45, 2006, the contents of which are alsoincorporated herein by reference. The EROD assay and Promega P450-Glo™Assay with selective and non-selective CYP1 inhibitors were used toconfirm that the CHO cell lines referred to above were expressing theexpected CYP1 enzymes in a functional form.

In the absence of inhibitors, CHO/CYP1A1 and CHO/CYP1B1 (but notwild-type CHO cells) converted 7-ethoxyresorufin to resorufin orLuciferin-CEE to luciferin, thereby confirming functional CYP1expression in these cells (see Table 1 below).

As expected, addition of the broad-spectrum CYP1 inhibitor,α-naphthoflavone, abolished activity in both CYP1 expressing cell lines(see Table 1 below). The selective inhibitor, tetramethoxystibene, is30-fold selective for CYP1B1 over CYP1A1 (Chun Y J, Kim S, Kim D, Lee SK and Guengerich F P, “A New Selective and Potent Inhibitor of HumanCytochrome P450 1B1 and its Application to Antimutagenesis”, Cancer Res61(22): 8164-70, 2001). Tetramethoxystilbene abolished activity at highconcentrations in both CYP1 expressing cell lines and preferentiallydecreased activity in CYP1B1 expressing cells (compared with CYP1A1expressing cells) cells at lower concentrations (see Table 1 below).

These results provide independent confirmation that the CYP1A1 andCYP1B1 expression levels are as expected.

Determining Cytotoxicity IC₅₀ Values in CHO, CHO/CYP1A1 and CHO/CYP1B1Cell Lines

A single cell suspension of CHO, CHO/CYP1A1 or CHO/CYP1B1 in 100 μl ofthe required cell culture medium was seeded onto 96-well plates at acell density of 1500 cells per well and placed in an incubator for 24 hat 37° C. The stock solution of test compound in DMSO was then added togive a concentration range of 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01,0.003, 0.001, 0 μM. The final concentration of DMSO 0.2% was found notto affect the growth characteristics of the various CHO cell lines. Thecells were incubated with the test compound for 72 or 96 h after whichall the medium was aspirated and replaced with 100 μl of fresh medium tocompensate for the loss of medium due to evaporation. The cells wereincubated with 20 μl of the MTS assay reagent for 1.5 h and theabsorbance per well at 510 nm measured using a plate reader. The meanabsorbance and standard deviation for each test compound concentrationwas calculated versus a series of controls including (a) cells plusmedium, (b) cell plus medium containing DMSO 0.2%, (c) medium alone, and(d) medium containing DMSO 0.2% and a range of test compoundconcentrations from 0 to 100 μmol dm⁻³. The cytotoxicity IC₅₀ value wascalculated from the plot of the percentage cell growth (where 100% cellgrowth corresponds to untreated control cells) versus test compoundconcentration.

Cytotoxicity IC₅₀ values are defined herein as the concentration ofcompound which kills 50% of cells and fold selectivity is calculated bydividing the IC₅₀ in non-CYP1 expressing cells with the IC₅₀ in CYP1A1or CYP1B1 expressing cells. Differential cytotoxicity IC₅₀ ratios arecalculated from compound IC₅₀ in normal CHO cells divided by IC₅₀ inCYP1A1 or CYP1B1 transfected CHO cells.

Promega™ CellTiter 96® Aqueous Non-Radioactive Cell Proliferation (MTS)Assay

The commercially available MTS assay is a homogeneous, colorimetricmethod for determining the number of viable cells in proliferation,cytotoxicity or chemosensitivity assays. The assay is composed ofsolutions of tetrazolium compound[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS] and an electron coupling reagent (phenazinemethosulfate) PMS. MTS is bioreduced by cells into a formazan productthat is soluble in tissue culture medium. The absorbance of the formazanproduct at 510 nm can be directly measured from 96-well assay plates.The quantity of formazan product as measured by the amount of absorbanceat 490 or 510 nm is directly proportional to the number of living cellsin culture.

Two compounds of the invention (SU025-04 and SU046-04), are designed torelease the phosphoramidate mustardsN,N-bis(2-chloro-ethyl)phosphoramide (CI-IPM) andN,N-bis(2-bromo-ethyl)phosphoramide (Br-IPM), respectively, whenactivated by CYP1B1. The high toxicities of the two phosphoramidatemustards CI-IPM and Br-IPM are significantly reduced when incorporatedin the prodrugs SU025-04 and SU046-04, respectively. Both SU025-04 andSU046-04 have cytotoxicity IC₅₀ values no less than 10 μmol dm⁻³ inwild-type CHO cells at 72 or 96 h exposure in marked contrast to CI-IPMand Br-IPM which have cytotoxicity IC₅₀ values below 0.007 μmol dm⁻³ inwild-type CHO cells after a 72 h exposure (see Table 2 below). Themechanism of activation of the two prodrugs can be deduced from theircomparative cytotoxicity IC₅₀ values in CHO-wild-type (which lacks CYP1enzyme expression), CHO/1A1, and CHO/CYP1B1 cells. For example, SU025and SU046 exhibit low toxicity in wild-type CHO cells but are highlytoxic to CHO/1B1 cells giving differential cytotoxicity IC₅₀ ratios of1689 and 5075, respectively at 72 h exposure. At a longer exposure timeof 96 h the CYP1B1-selective prodrugs SU025-04 and SU046-04 are 3367 and5400-fold more toxic to CYP1B1 expressing cells than non-CYP1B1expressing cells (see Table 2 below). Compounds SU025-04 and SU046-04are therefore demonstrably CYP1B1-activated prodrugs. SU025-04 andSU046-04 exhibit similarly low cytotoxicity to wild-type CHO andCHO/CYP1A1 cells with differential cytotoxicity IC₅₀ ratios <1 at 72 hexposure indicating that the highly toxic phosphoramidate mustards arenot released by CYP1A1 activation (see Table 2 below). As expected fromthe literature the two clinically used prodrugs ifosfamide andcyclophosphamide which also generate alkylating isophosphoamidatemustards when activated by CYP2B6 and CYP3A4 but not CYP1 enzymes (e.g.:McFadyen M C, Melvin W T and Murray G I, “Cytochrome P450 Enzymes: NovelOptions for Cancer Therapeutics”, Mol Cancer Ther., 3(3): 363-71, 2004)are both non-toxic at the highest concentration of 100 μmol dm⁻³ andlongest 96 h exposure times used in this cytotoxicity assay (see againTable 2 below).

Example 4: Prodrug Cytotoxicity in Primary Human Tumour Cell Lines

Prodrug Cytotoxicity in a Primary Human Head and Neck Squamous CellCarcinoma Tumour Cell Line (UT-SCC-14) which Constitutively ExpressesCYP1B1

Greer, et al., in Proc. Am. Assoc. Cancer Res., 45: 3701, 2004, reportedthat CYP1B1 was over-expressed during the malignant progression of headand neck squamous cell carcinoma (HNSCC) but not in normal epithelium. Aprimary UT-SCC-14 tumour cell line was isolated from a cancer patientwith HNSCC (see e.g. Yaromina et. al., Radiother Oncol., 83: 304-10,2007 and Hessel et al., Int J Radiat Biol., 80; 719-27, 2004. Thepatient was a male, aged 25, with an HNSCC characterized by thefollowing clinicopathological parameters: location, scc linguae; T₃ N₁,M₀; site, tongue; lesion, primary; grade G2. The UT-SCC-14 cell lineconstitutively expresses CYP1B1 at the mRNA and protein level and wasused to demonstrate compound cytotoxicity in cancer cell derived from ahuman cancer characterised by over-expression of CYP1B1 (Greer, et al.,in Proc. Am. Assoc. Cancer Res., 45: 3701, 2004).

UT-SCC-14 tumour cells: The HNSCC cell line was grown under standardcell culture conditions in EMEM (500 ml) supplemented with foetal calfserum (50 ml), non-essential amino acids (100×, 5 ml), sodium pyruvate(100 mmol dm⁻³, 5 ml), L-glutamine (200 mmol dm⁻³, 5 ml) with penicillin100 IU/ml/streptomycin (100 μg/ml, 5 ml) according to literature methods(Hessel et al., Int J Radiat Biol., 80; 719-27, 2004, the contents ofwhich are incorporated herein by reference).

Determining Prodrug Cytotoxicity IC₅₀ Values in Primary Nead and NeckTumour Cell Lines

A UT-SCC-14 tumour cell suspension at 2000 cells per well on a 96-wellplate and if necessary add fresh media to give a total volume per wellof 100 μl. The cells were allowed to attach for 4 h in an incubator.After 4 h it was confirmed that the cells had adhered to the bottom ofthe 96-well plate under a microscope, then the medium was removed andreplaced with fresh medium containing a stock solution of the testcompound in ethanol to give the following final concentrations 0, 0.001,0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 μmol dm⁻³ at a finalvolume of 100 μl per well. The final concentration of ethanol 0.2% wasfound not to effect the growth characteristics of the UT-SCC-14 cellline. The UT-SCC-14 cells were incubated with test compound for 72 hafter which time all aspirated and replaced with 100 μl of fresh mediumto compensate for the loss of medium due to evaporation. The cells wereincubated with 20 μl of the MTS assay reagent for 1.5 h and theabsorbance per well at 510 nm measured using a plate reader. The meanabsorbance and standard deviation for each test compound concentrationwas calculated versus a series of controls including (a) cells plusmedium, (b) cell plus medium containing ethanol 0.2%, (c) medium alone,and (d) medium containing ethanol 0.2% and a range of test compoundconcentrations from 0 to 100 μmol dm⁻³. The cytotoxicity IC₅₀ value wascalculated from the plot of the percentage cell growth (where 100% cellgrowth corresponds to untreated control cells) versus test compoundconcentration.

Cytotoxicity IC₅₀ values are defined herein as the concentration ofcompound which kills 50% of the UT-SCC-14 tumour cells. The commerciallyavailable MTS assay is a homogeneous, colorimetric method fordetermining the number of viable cells in proliferation, cytotoxicity orchemosensitivity assays and was used as described previously in thisExample 3 above.

Two compounds of the invention (SU025-04 and SU046-04), are designed torelease the phosphoramidate mustardsN,N-bis(2-chloro-ethyl)phosphoramide mustard (CI-IPM) andN,N-bis(2-bromo-ethyl)phosphoramide mustard (Br-IPM), respectively, whenactivated by CYP1B1. The cytotoxicity IC₅₀ values for SU025-04 andSU046-04 in the UT-SCC-14 tumour cells after 72 h exposure were0.05±0.01 μmol dm⁻³ and 0.02±0.01 μmol dm⁻³, respectively. The data showthe potent cytotoxicity of SU025-04 and SU046-04 in the UT-SCC14 cellline from a cancer patient with HNSCC which over-expresses CYP1B1.

SU025-04 and SU046-04 were evaluated in 3 additional primary head andneck cell lines including the UT-SCC-8, the UT-SCC-9 and the UTSCC-10cultured under the same conditions as the UT-SCC-14. For SU025-04 thecytotoxicity IC₅₀ in μmol dm⁻³ were UT-SCC-8 (0.31±0.06), UT-SCC-9(0.43±0.07), UT-SCC-10 (0.22±0.03) after a 72 h exposure. For SU025-04the cytotoxicity IC₅₀ in μmol dm⁻³ were UT-SCC-8 (0.06±0.02), UT-SCC-9(0.15±0.02), UT-SCC-10 (0.09±0.03) after a 72 h exposure. The dataindicate that SU046-04 is a more potent cytotoxin than SU025-04 across arange of primary head and neck cell lines which contitutively expressCYP1B1.

One compound of the invention, SU037-04, was designed to releasecamptothecin when activated by CYP1B1. For SU037-04 the cytotoxicityIC₅₀ in mol dm⁻³ for each primary tumour cell line was UT-SCC-8(0.56±0.04), UT-SCC-9 (0.22±0.08), UT-SCC-10 (0.21±0.04), UT-SCC-14(0.12±0.07) after a 72 h exposure.

One compound of the invention, SU048-04, was designed to releasegemcitabine when activated by CYP1B1. The cytotoxicity IC₅₀ for SU048-04in the UT-SCC-14 tumour cell line was 0.94±0.02 μmol dm⁻³ after a 72 hexposure. Co-incubation with α-napthoflavone (a potent CYP1B1 inhibitor)at 10 μmol dm⁻³ significantly reduced the toxicity of SU048-04 to12.2±0.2 μmol dm⁻³ thereby providing indirect evidence for theactivation of the prodrug by CYP1B1 constitutively expressed in thecells.

Example 5: Anti-Tumour Activity of SU046-04 in a Primary Human TumourXenograft Model which Constitutively Expresses CYP1B1

Primary UTSCC-14 cell lines 3×10⁶ were implanted subcutaneously in theflank of nude mice. Mice were randomized to 10 animals per group whenthe tumour volume was 100 to 150 mm³. SU046-04 was givenintraperitoneally at 12, 25 and 50 mg/Kg in PBS versus vehicle alone for2 cycles: daily for 5 days/2 days off. Tumour volume was measured every4 days using calipers. A significant inhibition of tumour growth wasobserved in all three treatment arms compared to the vehicle alone.Tumour growth delay at 28 days was 31% at 12 mg/Kg, 56% at 25 mg/Kg, and90% at 50 mg/Kg with 4/10 complete responses. No observed adverseeffects or significant body weight loss after highest exposure 250mg/Kg.

TABLE 1 Specific CYP1 enzyme activity in Chinese Hamster Ovary (CHO)cells stably transfected with CYP1A1 or CYP1B1 determined using bothfluorogenic and luminogenic substrates ^(a)Specific activity/pmolresorufin or luciferin min⁻¹ mg protein⁻¹ CHO CHO/CYP1A1 CHO/CYP1B1ethoxyresorufin Luciferin-CEE ethoxyresorufin Luciferin-CEEethoxyresorufin Luciferin-CEE nd nd 29 ± 4 21 ± 3 19 ± 3 22 ± 4 chemicalinhibitor ^(b)α-naphthoflavone 10 μmol dm⁻³ nd nd nd nd nd nd ^(c)TMS 5μmol dm⁻³ nd nd nd nd nd nd 10 nmol dm⁻³ nd nd 26 ± 4 20 ± 5 5 ± 2 3 ± 2^(a)Measured by two methods including the fluoresecent 7-ethoxyresorufinO-deethylation (EROD) assay or the Promega P450-Glo ™ Assay utilizingLuciferin-CEE as a luminogenic substrate for CYP1 enzymes. Luciferin-CEEfor CYP1A1 K_(m)app ~30 μmol dm⁻³ Luciferin-CEE for CYP1B1 K_(m)app ~20μmol dm⁻³ Ethoxyresorufin for CYP1A1and CYP1B1 K_(m) ~0.27 μmol dm⁻³ nd= no detectable activity. ^(b)α-naphthoflavone inhibits all CYP1 enzymesat 10 μmol dm⁻³. ^(c)Tetramethoxystilbene (TMS) is a selective inhibitorof CYP1B1 with IC₅₀ of  6 nmol dm⁻³ 30-fold greater than CYP1A1 andinhibits both enzymes at >1 μmol dm⁻³.

TABLE 2 In vitro cytotoxicity (IC₅₀) of prodrugs in a Chinese HamsterOvary (CHO) cell line stably transfected with CYP1A1 or CYP1B1 comparedto isophosphoramide mustard (IPM), cyclophosphamide, and ifosfamide.^(a,b,c)Cytotoxicity (IC₅₀)/μmol dm⁻³ CHO CHO/CYP1B1 IC₅₀ ratioCHO/CYP1A1 IC₅₀ ratio CHO CHO/CYP1B1 IC₅₀ ratio Compound 72 h 72 h 72 h72 h 72 h 96 h 96 h 96 h SU025-04  15.2 ± 0.2   0.009 ± 0.005 1689  25.5± 1.0   <1 10.1 ± 0.3 0.003 ± 0.002 3367 CI-IPM 0.007 ± 0.004 0.008 ±0.005 <1 0.006 ± 0.002 1.2 SU046-04  20.3 ± 1.2   0.004 ± 0.002 5075 20.3 ± 2.1   <1 16.2 ± 0.2 0.003 ± 0.001 5400 Br-IPM 0.005 ± 0.0020.004 ± 0.002 1.25 0.005 ± 0.003 1 ifosfamide ND ND ND ND ND ND ND NDcyclophosphamide ND ND ND ND ND ND ND ND ^(a)Cytotoxicity measured usingthe Promega CellTiter 96 ® AQ_(ueous) Non-Radioactive Cell ProliferationAssay. ^(b)Exposure time was 72 or 96 h, dose range 0 to 100 μmol dm⁻³.^(c)IC₅₀ ratios calculated from compound IC₅₀ in normal CHO cellsdivided by IC₅₀ in CYP1A1 or CYP1B1 transfected CHO cells. ND = notdetectable, indicating <50% toxicity observed at highest compoundconcentration tested.

TABLE 3 Substituent effect on the fragmentation of benzofuran ether andcarbamate linked coumarins by CYP1 enzymes and human liver microsomes(HLM).

^(a,b)Specific fragmentation activity/pmol coumarin m⁻¹ pmol cytochromeP450⁻¹ Compound Structure Substituent CYP1A1 CYP1A2 CYP1B1 HLM SU010A AR⁵ = H; R⁷ = H 20.91 ± 00.34  8.66 ± 0.28  5.19 ± 0.15 22.8 ± 0.69VG015-05 A R⁵ = F; R⁷ = H 19.35 ± 3.08 nd  3.16 ± 0.28 nd VG016-05 A R⁵= F; R⁷ = F no release no release no release no release VG017-05 A R⁵ =F; R⁷ = Me  5.39 ± 1.34 nd  2.95 ± 0.59 nd VG027-05 A R⁵ = MeO; R⁷ = H88.10 ± 5.69 20.93 ± 0.59 11.83 ± 0.15  8.98 ± 0.49 VG029-05 A R⁵ = H;R⁷ = MeO 19.53 ± 1.07 nd  6.45 ± 1.15 nd VG035-04 A R⁵ = Br; R⁷ = H28.08 ± 1.98 nd  8.85 ± 0.28 nd VG028-05 A R⁵ = Cl; R⁷ = H 17.52 ± 0.83nd  6.67 ± 0.01 nd VG035-05 A R⁵ = MeO; R⁷ = MeO no release no release13.65 ± 1.00 no release SU018-03 B R⁵ = H; R⁷ = H  2.41 ± 0.18 norelease no release no release VG032-05 B R⁴ = MeO; R⁷ = H 12.19 ± 3.02no release  1.53 ± 0.09  3.21 ± 0.25 VG036-05 B R⁵ = Br; R⁷ = H norelease no release no release no release VG041-05 B R⁵ = MeO; R⁷ = MeO 5.51 ± 0.06 no release no release no release ^(a)Ether or carbamatelinker fragmentation was monitored by coumarin anion release by kineticfluorimetry using excitation/emission wavelengths: λex = 350 nm/λem =450 nm. CYP1 enzyme concentration was 10 pmol and the volume of HLM was60 μl a final reaction volume of 1.5 ml. Specific fragmentationactivities are quoted as the mean ± standard deviation of threemeasurements. nd = not determined.

1. A compound of formula (I):

(wherein: X¹ is such that —X¹—X² is —O—X², —S—X², —SO₂—O—X²,—SO₂NZ¹⁰—X², conjugated alkenemethyloxy or conjugated alkenemethylthio,conjugated alkenemethylSO₂—O, conjugated alkenemethyl-SO₂NZ¹⁰ or of theformula:

—X² is absent or is such that X¹—X²-Effector is one of

each n and m is independently 0 or 1; p is 0, 1 or 2; X³ is oxygen orsulfur and additionally, when m=0, may be SO₂—O, SO₂NZ¹⁰, conjugatedalkenemethyloxy, conjugated alkenemethylthio, conjugatedalkenemethyl-SO₂—O or conjugated alkenemethyl-SO₂NZ¹⁰ each of Y¹, Y² andY³ is independently carbon or nitrogen, wherein if Y¹ is nitrogen, Z¹ isabsent, if Y² is nitrogen, Z³ is absent and if Y³ is nitrogen, Z⁵ isabsent; Y⁴ is an oxygen, carbon or nitrogen atom, sulfoxide or sulfone;—Y⁵— is either (i) a single bond, (ii) ═CH—, wherein the double bond =in ═CH— is connected to Y⁴, or (iii) —CH₂— or —CH₂CH₂—, or one of (ii)to (iii) wherein the hydrogen atom in (ii) is or one or more hydrogenatoms in (iii) are replaced with a substituent Z¹¹, wherein Z¹¹ isselected independently from alkyl, alkenyl, alkynyl, aryl, aralkyl,alkyloxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy, alkylthioxy,alkenylthioxy, alkynylthioxy, arylthioxy, aralkylthioxy, amino, hydroxy,thio, halo, carboxy, formyl, nitro and cyano; each of Z¹—Z⁴, wherepresent, are independently selected from hydrogen, alkyl, alkenyl,alkynyl, aryl, aralkyl, alkyloxy, alkenyloxy, alkynyloxy, aryloxy,aralkyloxy, alkylthioxy, alkenylthioxy, alkynylthioxy, arylthioxy,aralkylthioxy, amino, hydroxy, thio, halo, carboxy, formyl, nitro andcyano; and Z⁵, where present, is independently selected from hydrogenalkyl, alkenyl, alkynyl, aryl, aralkyl, alkyloxy, alkenyloxy,alkynyloxy, aryloxy, aralkyloxy, alkylthioxy, alkenylthioxy,alkynylthioxy, arylthioxy, aralkylthioxy, amino, hydroxy, thio, carboxy,formyl, nitro and cyano, or one of Z² & Z³, Z³ & Z⁴ and Z⁴ and Z⁵together with the atoms to which they are connected form an aromaticring fused to the remainder of the compound, provided that at least oneof Z¹, Z² and Z⁴ is hydrogen; Z⁶ is selected from hydrogen, alkyl,alkenyl, alkynyl, aryl and aralkyl; none, one or two of Y⁶ may benitrogen atoms with the remainder being carbon atoms; each Z⁷ isindependently hydrogen, alkyl or aryl; each Z⁸ is independently selectedfrom hydrogen, an electron withdrawing group, unsubstituted C₁-C₆ alkyl,substituted C₁-C₆ alkyl, unsubstituted C₁-C₆ alkoxy, and substitutedC₁-C₆ alkoxy where the substituted alkyl or alkoxy are substituted withone or more groups selected from ether, amino, mono- or di-substitutedamino, cyclic C₁-C₅ alkylamino, imidazolyl, C₁-C₆ alkylpiperazinyl,morpholino, thiol, thioether, tetrazole, carboxylic acid, ester, amido,mono- or di-substituted amido, N-connected amide, N-connectedsulfonamide, sulfoxy, sulfonate, sulfonyl, sulfoxy, sulfinate, sufinyl,phosphonooxy, phosphate and sulfonamide; each Z⁹ is independently oxygenor sulfur; Z¹⁰ is hydrogen or alkyl, for example a C₁₋₄ alkyl; Effectoris a molecule having a pharmacological or diagnostic function), or apharmaceutically acceptable salt, ester, amide or solvate thereof.